Proteases, nucleic acids encoding them and methods for making and using them

ABSTRACT

The invention is directed to polypeptides having protease activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of diagnostic, therapeutic, and industrial contexts. The polypeptides of the invention can be used as, e.g., an additive for a detergent, for processing foods and for chemical synthesis utilizing a reverse reaction. Additionally, the polypeptides of the invention can be used in food processing, brewing, bath additives, alcohol production, peptide synthesis, enantioselectivity, hide preparation in the leather industry, waste management and animal degradation, silver recovery in the photographic industry, medical treatment, silk degumming, biofilm degradation, biomass conversion to ethanol diodefense, antimicrobial agents and disinfectants, personal care and cosmetics, biotech reagents, in increasing starch yield from corn wet milling and pharmaceuticals such as digestive aids and anti-inflammatory (anti-phlogistic) agents.

TECHNICAL FIELD

This invention relates to molecular and cellular biology andbiochemistry. In particular, the invention relates to protease enzymes,polynucleotides encoding the enzymes, methods of making and using thesepolynucleotides and polypeptides. The polypeptides of the invention canbe used in a variety of diagnostic, therapeutic, and industrialcontexts. The polypeptides of the invention can be used as, e.g., anadditive for a detergent, for processing foods and for chemicalsynthesis utilizing a reverse reaction. Additionally, the polypeptidesof the invention can be used in food processing, brewing, bathadditives, alcohol production, peptide synthesis, enantioselectivity,hide preparation in the leather industry, waste management and animaldegradation, silver recovery in the photographic industry, medicaltreatment, silk degumming, biofilm degradation, biomass conversion toethanol, biodefense, antimicrobial agents and disinfectants, personalcare and cosmetics, biotech reagents, in increasing starch yield fromcorn wet milling and pharmaceuticals such as digestive aids andanti-inflammatory (anti-phlogistic) agents.

BACKGROUND

Enzymes are used within a wide range of applications in industry,research, and medicine. Through the use of enzymes, industrial processescan be carried out at reduced temperatures and pressures and with lessdependence on the use of corrosive or toxic substances. The use ofenzymes can thus reduce production costs, energy consumption, andpollution as compared to non-enzymatic products and processes. Animportant group of enzymes is the proteases. Proteases are carbonylhydrolases which generally act to cleave peptide bonds of proteins orpeptides. Proteolytic enzymes are ubiquitous in occurrence, found in allliving organisms, and are essential for cell growth and differentiation.The extracellular proteases are of commercial value and find multipleapplications in various industrial sectors. Industrial applications ofproteases include food processing, brewing, alcohol production, peptidesynthesis, enantioselectivity, hide preparation in the leather industry,waste management and animal degradation, silver recovery in thephotographic industry, medical treatment, silk degumming, biofilmdegradation, biomass conversion to ethanol, biodefense, antimicrobialagents and disinfectants, personal care and cosmetics, biotech reagentsand in increasing starch yield from corn wet milling. Additionally,proteases are important components of laundry detergents and otherproducts. Within biological research, proteases are used in purificationprocesses to degrade unwanted proteins. It is often desirable to employproteases of low specificity or mixtures of more specific proteases toobtain the necessary degree of degradation.

Proteases are classified according to their catalytic mechanisms. TheInternational Union of Biochemistry and Molecular Biology (IUBMB)recognizes four mechanistic classes: (1) the serine proteases; (2) thecysteine proteases; (3) the aspartic proteases; and (4) themetalloproteases. In addition, the IUBMB recognizes a class ofendopeptidases (oligopeptidases) of unknown catalytic mechanism.Classification by catalytic types has been suggested to be extended by aclassification by families based on the evolutionary relationships ofproteases (see, e.g., Rawlings, N. D. and Barett, A. J., (1993),Biochem. J., 290, 205-218). The serine proteases have alkaline pHoptima, the metalloproteases are optimally active around neutrality, andthe cysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks. Bacilluis. vol. 2. edited by Harwood, 1989 Plenum Press, NewYork). Aspartic proteases are rare for bacteria and to date none havebeen reported for bacterial pathogens. Metalloproteases, on the otherhand, seem to be a common feature in most bacterial pathogens. Thus,basic two classes of bacterial proteases are serine proteases andmetalloproteases.

Serine proteases are characterized by a catalytic triad of serine,histidine, and aspartic acid residues. They include a diverse class ofenzymes having a wide range of specificities and biological functions.The serine proteases class comprises two distinct families: thechymotrypsin family, which includes the mammalian enzymes such aschymotrypsin, trypsin, elastase, or kallikrein, and the subtilisinfamily, which include the bacterial enzymes such as subtilisin. Thegeneral 3D structure is different in two families, but they have thesame active site geometry and catalysis proceeds via the same mechanism.Serine proteases are used for a variety of industrial purposes. Forexample, the serine protease subtilisin is used in laundry detergents toaid in the removal of proteinaceous stains (e.g., Crabb, ACS SymposiumSeries 460:82-94, 1991). In the food processing industry, serineproteases are used to produce protein-rich concentrates from fish andlivestock, and in the preparation of dairy products (Kida et al.,Journal of Fermentation and Bioengineering 80:478-484, 1995; Haard andSimpson, in Martin, A. M., ed., Fisheries Processing: BiotechnologicalApplications, Chapman and Hall, London, 1994, 132-154; Bos et al.,European Patent Office Publication 494 149 A1).

Metalloproteases (MPs) and serine proteases form the most diverse of thecatalytic types of proteases. They can be found in bacteria, fungi, aswell as in higher organisms. They differ widely in their sequences andstructures, but the great majority of enzymes contain a zinc atom whichis catalytically active. In some cases, zinc may be replaced by anothermetal such as cobalt or nickel without loss of activity. The catalyticmechanism leads to the formation of a non-covalent tetrahedralintermediate after the attack of a zinc-bound water molecule on thecarbonyl group of the scissile bond. This intermediate is furtherdecomposed by transfer of the glutamic acid portion to the leavinggroup.

In general, enzymes, including proteases, are active over a narrow rangeof environmental conditions (temperature, pH, etc.), and many are highlyspecific for particular substrates. The narrow range of activity for agiven enzyme limits its applicability and creates a need for a selectionof enzymes that (a) have similar activities but are active underdifferent conditions or (b) have different substrates. For instance, anenzyme capable of catalyzing a reaction at 50° C. may be so inefficientat 35° C., that its use at the lower temperature will not be feasible.For this reason, laundry detergents generally contain a selection ofproteolytic enzymes, allowing the detergent to be used over a broadrange of wash temperature and pH. In view of the specificity ofproteolytic enzymes and the growing use of proteases in industry,research, and medicine, there is an ongoing need in the art for newenzymes and new enzyme inhibitors.

SUMMARY

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1; SEQ ID NO:3;SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ IDNO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ IDNO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ IDNO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ IDNO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ IDNO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ IDNO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ IDNO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ IDNO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ IDNO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123;SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ IDNO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158;SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ IDNO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248and/or SEQ ID NO:254, over a region of at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400,2450, 2500, or more residues, encodes at least one polypeptide having aprotease activity, and the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.

Exemplary nucleic acids of the invention also include isolated orrecombinant nucleic acids encoding a polypeptide having a sequence asset forth in SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ IDNO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ IDNO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ IDNO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ IDNO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ IDNO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ IDNO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ IDNO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ IDNO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ IDNO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ IDNO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118;SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ IDNO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147;SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ IDNO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235;SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or a polypeptide encodedby SEQ ID NO:145, and subsequences thereof and variants thereof. In oneaspect, the polypeptide has a protease activity.

The following list summarizes polypeptide sequence and nucleic acidcoding sequence relationships between exemplary sequences of theinvention; for examples, SEQ ID NO:2 is encoded by SEQ ID NO:1, SEQ IDNO:255 is encoded by SEQ ID NO:254, etc.). DNA SEQ Protein SEQ ID IDNOS: NOS: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2425 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 4849 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 7273 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 9697 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133134 135 136 137 138 139 140 141 142 143 144 145 N/A 146 147 150 151 158159 164 165 171 172 179 180 187 188 193 194 199 200 204 205 210 211 218219 222 223 229 230 234 235 241 242 248 249 254 255

In one aspect, the invention also provides proteases, andprotease-encoding nucleic acids, with a common novelty in that they wereinitially isolated/derived from mixed cultures. The invention providesprotease-encoding nucleic acids isolated from mixed cultures comprisinga nucleic acid sequence having at least about 10, 15, 20, 25, 30, 35,40, 45, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary nucleic acid of the invention, e.g.,SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ IDNO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ IDNO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ IDNO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ IDNO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ IDNO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ IDNO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ IDNO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ IDNO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ IDNO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ IDNO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; SEQ ID NO:109; SEQID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:119;SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ IDNO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:146;SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ IDNO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234;SEQ ID NO:241; SEQ ID NO:248 and/or SEQ ID NO:254, over a region of atleast about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or more.

In one aspect, the invention also provides proteases, andprotease-encoding nucleic acids, with a common novelty in that wereinitially derived from a common source, e.g., an archeal source, abacterial source, a fungal source (e.g., filamentous ascomycetes, suchas Cochliobolus heterostrophus, e.g., C. heterostrophus strain C4,having ATCC accession no. 48331), or an environmental source, e.g., amixed environmental source, e.g., as set forth below. SEQ ID NO: Source1, 2 Archea 17, 18 Archea 63, 64 Archea 15, 16 Bacteria 13, 14 Bacteria5, 6 Bacteria 3, 4 Bacteria 57, 58 Bacteria 7, 8 Bacteria

187, 188Cochliobolus heterostrophus strain C4 (ATCC 48331)

210, 211 Cochliobolus heterostrophus strain C4 (ATCC 48331)

234, 235 Cochliobolus heterostrophus strain C4 (ATCC 48331)

164, 165 Cochliobolus heterostrophus strain C4 (ATCC 48331)

199, 200 Cochliobolus heterostrophus strain C4 (ATCC 48331)

229, 230 Cochliobolus heterostrophus strain C4 (ATCC 48331)

158, 159 Cochliobolus heterostrophus strain C4 (ATCC 48331)

193, 194 Cochliobolus heterostrophus strain C4 (ATCC 48331)

222, 223Cochliobolus heterostrophus strain C4 (ATCC 48331)

179, 180 Cochliobolus heterostrophus strain C4 (ATCC 48331)

218, 219Cochliobolus heterostrophus strain C4 (ATCC 48331)

150, 151 Cochliobolus heterostrophus strain C4 (ATCC 48331)

171, 172Cochliobolus heterostrophus strain C4 (ATCC 48331)

204, 205Cochliobolus heterostrophus strain C4 (ATCC 48331)

254, 255Cochliobolus heterostrophus strain C4 (ATCC 48331)

248, 249 Cochliobolus heterostrophus strain C4 (ATCC 48331)

241, 242 Cochliobolus heterostrophus strain C4 (ATCC 48331) 85, 86Environmental 11, 12 Environmental 121, 122 Environmental 117, 118Environmental 119, 120 Environmental 83, 84 Environmental  9, 10Environmental 93, 94 Environmental 101, 102 Environmental 127, 128Environmental 129, 130 Environmental 139, 140 Environmental 146, 147Environmental 33, 34 Environmental 113, 114 Environmental 39, 40Environmental 71, 72 Environmental 133, 134 Environmental 45, 46Environmental 77, 78 Environmental 19, 20 Environmental 59, 60Environmental 41, 42 Environmental 111, 112 Environmental 123, 124Environmental 125, 126 Environmental 107, 108 Environmental 109, 110Environmental 79, 80 Environmental 23, 24 Environmental 27, 28Environmental 143, 144 Environmental 69, 70 Environmental 141, 142Environmental 55, 56 Environmental 61, 62 Environmental 73, 74Environmental 87, 88 Environmental 37, 38 Environmental 47, 48Environmental 51, 52 Environmental 65, 66 Environmental 29, 30Environmental 67, 68 Environmental 25, 26 Environmental 75, 76Environmental 81, 82 Environmental 31, 32 Environmental 35, 36Environmental 43, 44 Environmental 49, 50 Environmental 137, 138Environmental 131, 132 Environmental 95, 96 Environmental 103, 104Environmental 135, 136 Environmental 145 Environmental 105, 106Environmental  99, 100 Environmental 97, 98 Environmental 89, 90Environmental 91, 92 Environmental 21, 22 Environmental 115, 116Environmental 53, 54 Environmental

For example (referring to the above list), the proteases, andprotease-encoding nucleic acids, as set forth in SEQ ID NO:2 (encoded bySEQ ID NO:1), SEQ ID NO:18 (encoded by SEQ ID NO:17), SEQ ID NO:64(encoded by SEQ ID NO:63) and SEQ ID NO:16 (encoded by SEQ ID NO:15)have a common novelty in that were initially derived from an archealsource, etc. with polypeptides and nucleic acids initially derived frombacterial, fungal (Cochliobolus heterostrophus), or environmentalsources.

In one aspect, the invention provides proteases, and protease-encodingnucleic acids, initially isolated/derived from environmental sources,e.g., mixed environmental sources, comprising a nucleic acid sequencehaving at least about 10, 15, 20, 25, 30, 35, 40, 45, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary nucleic acid of the invention over a region of at leastabout 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more,residues, wherein the nucleic acid encodes at least one polypeptidehaving a protease activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection.

Regarding proteases, and protease-encoding nucleic acids, of theinvention with a common novelty in that they were initially derived fromthe filamentous ascomycetes Cochliobolus heterostrophus, in one aspect,these polypeptides and nucleic acids were initially isolated by growingCochliobolus on either chicken feed or corn fiber, which was the solenitrogen source. Supernatant of the media was concentrated and run on agel. The resulting proteins isolated from the gel bands were analyzed bymass spectrometry. These proteins were sequenced and compared to theCochliobolus genomic sequence. The proteases, and protease-encodingnucleic acids, of the invention initially isolated from Cochliobolusheterostrophus are summarized as follows: SEQ ID NO: of protein sequenceSEQ ID NO: of full SEQ ID NO: of DNA of coding Protease gene (exons andSEQ ID NOS: of sequence of coding sequence (exons ID introns) exonsequences sequence (exons only) only) A 181 182-186 187 188 B 206207-209 210 211 C 231 232-233 234 235 D 160 161-163 164 165 E 195196-198 199 200 F 224 225-228 229 230 G 152 153-157 158 159 H 189190-192 193 194 I 220 221 222 223 J 173 174-178 179 180 K 212 213-217218 219 L 148 149 150 151 M 166 167-170 171 172 N 201 202-203 204 205 O250 251-253 254 255 P 243 244-247 248 249 Q 236 237-240 241 242

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d“nr pataa”−F F, and all other options are set to default.

Another aspect of the invention is an isolated or recombinant nucleicacid including at least 10 consecutive bases of a nucleic acid sequenceof the invention, sequences substantially identical thereto, and thesequences complementary thereto.

In one aspect, protease activity of the invention comprises catalyzinghydrolysis of peptide bonds. The term “protease activity” includeshydrolysis of any peptide bond, including protease activity, peptidaseactivity and/or proteinase activity. The protease activity can comprisean endoprotease activity and/or an exoprotease activity. The proteaseactivity can comprise a carboxypeptidase activity, an aminopeptidaseactivity, a serine protease activity, a metalloprotease activity (e.g.,matrix metalloprotease or collagenase activity), a cysteine proteaseactivity and/or an aspartic protease activity. In one aspect, proteaseactivity can comprise activity the same or similar to a chymotrypsin, atrypsin, an elastase, a kallikrein and/or a subtilisin activity. Theprotease activity can comprise a peptidase activity, such as adipeptidylpeptidase or a carboxypeptidase activity. In alternativeaspects, the protease activity can comprise a acrocylindropepsinactivity, acrosin activity, actinidain activity, acylaminoacyl-peptidaseactivity, ADAM 17 endopeptidase activity, ADAM10 endopeptidase activity,adamalysin activity, ADAMTS-4 endopeptidase activity, adenain activity,aeromonolysin activity, alanine carboxypeptidase activity, alpha-lyticendopeptidase activity, alternative-complement pathway C3/C5 convertaseactivity, aminopeptidase B activity, aminopeptidase Ey activity,aminopeptidase I activity, ananain activity, anthrax lethal factorendopeptidase activity, asclepain activity, aspartyl aminopeptidaseactivity, aspergillopepsin I activity, aspergillopepsin II activity,assemblin activity, astacin activity, atrolysin A activity, atrolysin Bactivity, atrolysin C activity, atrolysin E activity, atrolysin Factivity, atroxase activity, aureolysin activity, bacillolysin activity,bacterial leucyl aminopeptidase activity, barrierpepsin activity,Beta-Ala-His dipeptidase activity, Beta-aspartyl-peptidase, beta-lyticmetalloendopeptidase activity, bleomycin hydrolase activity,bontoxilysin activity, bothrolysin activity, bothropasin activity,brachyurin activity, calpain-1 activity, calpain-2 activity, cancerprocoagulant activity, candidapepsin activity, carboxypeptidase Aactivity, carboxypeptidase A2 activity, carboxypeptidase B activity,carboxypeptidase C activity, carboxypeptidase D activity,carboxypeptidase H activity, carboxypeptidase M activity,carboxypeptidase T activity, carboxypeptidase U activity, caricainactivity, caspase-1 activity, cathepsin B activity, cathepsin Dactivity, cathepsin E activity, cathepsin F activity, cathepsin Gactivity, cathepsin H activity, cathepsin K activity, cathepsin Lactivity, cathepsin O activity, cathepsin S activity, cathepsin Tactivity, cathepsin V activity, cerevisin activity, choriolysin Hactivity, choriolysin L activity, chymase activity, chymopapainactivity, chymosin activity, chymotrypsin activity (e.g., chymotrypsin Cactivity), classical-complement pathway C3/C5 convertase, clostridialaminopeptidase activity, clostripain activity, coagulation factor IXaactivity, coagulation factor VIIa activity, coagulation factor Xaactivity, coagulation factor XIa activity, coagulation factor XIIaactivity, coccolysin activity, complement component C1r activity,complement component C1s activity, complement factor D activity,complement factor I activity, cruzipain activity, cucumisin activity,cysteine-type carboxypeptidase activity, cystinyl aminopeptidaseactivity, cytosol alanyl aminopeptidase activity, cytosol nonspecificdipeptidase activity, dactylysin activity, deuterolysin activity,dipeptidase E activity, dipeptidyl-dipeptidase activity,dipeptidyl-peptidase I activity, dipeptidyl-peptidase II activity,dipeptidyl-peptidase III activity, dipeptidyl-peptidase IV activity,D-stereospecific aminopeptidase activity, endopeptidase Clp activity,endopeptidase La activity, endopeptidase So activity,endothelin-converting enzyme 1 activity, endothiapepsin activity,enteropeptidase activity, envelysin activity, fibrolase activity, ficainactivity, flavastacin activity, flavirin activity, fragilysin activity,fruit bromelain activity, furin activity, gametolysin activity,gamma-D-glutamyl-meso-diaminopimelate peptidase I activity,gamma-glutamyl hydrolase activity, gamma-renin activity, gastricsinactivity, gelatinase A activity, gelatinase B activity, gingipain Kactivity, gingipain R activity, Glu-Glu dipeptidase activity, glutamatecarboxypeptidase II activity, Glutamate carboxypeptidase activityGlutamyl aminopeptidase activity, Glutamyl endopeptidase II activity,Glutamyl endopeptidase activity, Glycyl endopeptidase activity, Gly-Xcarboxypeptidase activity, GPR endopeptidase activity, Granzyme Aactivity, Granzyme B activity, Helper-component proteinase activity,Hepacivirin activity, Histolysain activity, HIV-1 retropepsin activity,HIV-2 retropepsin activity, Horrilysin activity, Hypodermin C activity,IgA-specific metalloendopeptidase activity, IgA-specific serineendopeptidase activity, Insulysin activity, Interstitial collagenaseactivity, Jararhagin activity, Kexin activity, Lactocepin activity,Legumain activity, Leishmanolysin activity, Leucolysin activity, Leucylaminopeptidase activity, Leucyl endopeptidase activity, Leukocyteelastase activity, Limulus clotting enzyme activity, Limulus clottingfactor B activity, Limulus clotting factor C activity, L-peptidaseactivity, Lysine(arginine) carboxypeptidase activity, Lysosomal Pro-Xcarboxypeptidase activity, Lysostaphin activity, Lysyl aminopeptidaseactivity, Lysyl endopeptidase activity, Macrophage elastase activity,Magnolysin activity, Matrilysin activity, Memapsin 1, Memapsin 2,Membrane alanine aminopeptidase, Membrane dipeptidase, Membrane Pro-Xcarboxypeptidase, Membrane-type matrix metalloproteinase-1, Meprin A,Meprin B, Metallocarboxypeptidase D, Methionyl aminopeptidase, Metridin,Met-Xaa dipeptidase, Microbial collagenase, Mitochondrial intermediatepeptidase, Mitochondrial processing peptidase, Mucoropepsin, Mucrolysin,Muramoylpentapeptide carboxypeptidase, Muramoyltetrapeptidecarboxypeptidase, Mycolysin, Myeloblastin, Nardilysin, Neopenthesin,Neprilysin, Neurolysin, Neutrophil collagenase,N-formylmethionyl-peptidase, Nodavirus endopeptidase, Non-stereospecificdipeptidase, Nuclear-inclusion-a endopeptidase, Oligopeptidase A,Oligopeptidase B, Omptin, Ophiolysin, Oryzin, O-sialoglycoproteinendopeptidase, Pancreatic elastase II, Pancreatic elastase, Pancreaticendopeptidase E, Papain, Pappalysin-1, Penicillopepsin, PepBaminopeptidase, Pepsin A, Pepsin B, Peptidyl-Asp metalloendopeptidase,Peptidyl-dipeptidase A, Peptidyl-dipeptidase B, Peptidyl-dipeptidaseDcp, Peptidyl-glycinamidase, Peptidyl-Lys metalloendopeptidase,Phytepsin, Picornain 2A, Picornain 3C, Pitrilysin, Plasma kallikrein,Plasmepsin I, Plasmepsin II, Plasmin, Plasminogen activator Pla,Polyporopepsin, Prepilin peptidase, Procollagen C-endopeptidase,Procollagen N-endopeptidase, Prolyl aminopeptidase, Prolyloligopeptidase, Pro-opiomelanocortin converting enzyme, Proproteinconvertase 1, Proprotein convertase 2, Proteasome endopeptidase complex,Protein C (activated), Proteinase K, Pseudolysin, Pycnoporopepsin,Pyroglutamyl-peptidase I, Pyroglutamyl-peptidase II, Renin, RepressorlexA, Rhizopuspepsin, Rhodotorulapepsin, Ruberlysin, Russellysin, S2Pendopeptidase, Saccharolysin, Saccharopepsin, Scutelarin,Scytalidopepsin A activity, Scytalidopepsin B, Semenogelase, Separase,Serine-type D-Ala-D-Ala carboxypeptidase, Serralysin, Signal peptidaseI, Signal peptidase II, Snake venom factor V activator, Snapalysin,Spermosin, Staphopain, Ste24 endopeptidase, Stem bromelain,Streptogrisin A, Streptogrisin B, Streptopain, Stromelysin 1,Stromelysin 2, Subtilisin, Tentoxilysin, Thermitase, Thermolysin,Thermomycolin, Thermopsin, Thermostable carboxypeptidase 1, Thimetoligopeptidase, Thrombin activity, Tissue kallikrein activity, Togavirinactivity, T-plasminogen activator activity, Trimerelysin I activity,Trimerelysin II activity, Tripeptide aminopeptidase activity,Tripeptidyl-peptidase I activity, Tripeptidyl-peptidase II activity,Trypsin activity, Tryptase activity, Tryptophanyl aminopeptidaseactivity, Tubulinyl-Tyr carboxypeptidase activity, Ubiquitinyl hydrolaseI activity, II-plasminogen activator activity, V-cath endopeptidaseactivity, Venombin A activity, Venombin AB activity, Xaa-Arg dipeptidaseactivity, Xaa-His dipeptidase, activity Xaa-methyl-His dipeptidaseactivity, Xaa-Pro aminopeptidase activity, Xaa-Pro dipeptidase activity,Xaa-Pro dipeptidyl-peptidase activity, Xaa-Trp aminopeptidase activity,Yapsin 1 activity, Zinc D-Ala-D-Ala carboxypeptidase activity or acombination thereof.

Some alternative activities of exemplary polypeptides of the invention(for example, as listed above) were determined by experimental data, byhomology (sequence comparison) to other sequences, or by both sequencecomparison and experimental results. However, an exemplary species ofthe invention, or a genus of polypeptides based on an exemplarysequence, is not limited to any specific protease activity. Thus, inalternative, but not limiting aspects, a polypeptide having a sequenceas set forth in SEQ ID NO:2 (encoded by SEQ ID NO:1), can have analkaline protease activity; a polypeptide having a sequence as set forthin SEQ ID NO:4 (encoded by SEQ ID NO:3), can have a serine proteaseactivity; a polypeptide having a sequence as set forth in SEQ ID NO:6(encoded by SEQ ID NO:5), can have a peptidase activity; a polypeptidehaving a sequence as set forth in SEQ ID NO:22 (encoded by SEQ ID NO:21,can have a serine protease activity; a polypeptide having a sequence asset forth in SEQ ID NO:26 (encoded by SEQ ID NO:25, can have asubtilisin-like secreted protease activity; a polypeptide having asequence as set forth in SEQ ID NO:28 (encoded by SEQ ID NO:27), canhave a serine protease activity (e.g., an alkaline serine proteaseactivity); a polypeptide having a sequence as set forth in SEQ ID NO:36(encoded by SEQ ID NO:35), can have a serine protease activity (e.g., analkaline serine protease activity); a polypeptide having a sequence asset forth in SEQ ID NO:38 (encoded by SEQ ID NO:37), can have a serineprotease activity; a polypeptide having a sequence as set forth in SEQID NO:42 (encoded by SEQ ID NO:41), can have a serine protease activity(e.g., an extracellular alkaline serine protease 2 activity); apolypeptide having a sequence as set forth in SEQ ID NO:50 (encoded bySEQ ID NO:49), can have a serine protease activity (e.g., an alkalineserine protease activity); a polypeptide having a sequence as set forthin SEQ ID NO:58 (encoded by SEQ ID NO:57), can have a serine proteaseactivity; a polypeptide having a sequence as set forth in SEQ ID NO:68(encoded by SEQ ID NO:67), can have a serine protease activity (e.g., analkaline serine protease activity); a polypeptide having a sequence asset forth in SEQ ID NO:74 (encoded by SEQ ID NO:73), can have a serineprotease activity (e.g., an alkaline serine protease activity); apolypeptide having a sequence as set forth in SEQ ID NO:76 (encoded bySEQ ID NO:75), can have a serine protease activity (e.g., a cold-activeserine alkaline protease activity); a polypeptide having a sequence asset forth in SEQ ID NO:82 (encoded by SEQ ID NO:81), can have a serineprotease activity; a polypeptide having a sequence as set forth in SEQID NO:86 (encoded by SEQ ID NO:85), can have a protease II activity; apolypeptide having a sequence as set forth in SEQ ID NO:90 (encoded bySEQ ID NO:89), can have a serine metalloprotease activity; a polypeptidehaving a sequence as set forth in SEQ ID NO:92 (encoded by SEQ IDNO:91), can have a metalloprotease activity; a polypeptide having asequence as set forth in SEQ ID NO:96 (encoded by SEQ ID NO:95), canhave a serine protease activity (e.g., a cold-active serine alkalineprotease activity); a polypeptide having a sequence as set forth in SEQID NO:98 (encoded by SEQ ID NO:97), can have a peptidase activity; apolypeptide having a sequence as set forth in SEQ ID NO:100 (encoded bySEQ ID NO:99), can have a prohormone convertase activity; a polypeptidehaving a sequence as set forth in SEQ ID NO:106 (encoded by SEQ IDNO:105), can have a collagenase activity; a polypeptide having asequence as set forth in SEQ ID NO:112 (encoded by SEQ ID NO:111), canhave an alkaline serine protease II activity; a polypeptide having asequence as set forth in SEQ ID NO:114 (encoded by SEQ ID NO:113), canhave a serine proteinase activity; a polypeptide having a sequence asset forth in SEQ ID NO:120 (encoded by SEQ ID NO:119), can have asubtilisin-like proteinase activity; a polypeptide having a sequence asset forth in SEQ ID NO:128 (encoded by SEQ ID NO:127), can have a serineproteinase activity (e.g., serine protease A activity); a polypeptidehaving a sequence as set forth in SEQ ID NO:134 (encoded by SEQ IDNO:133), can have a leucine aminopeptidase activity; a polypeptidehaving a sequence as set forth in SEQ ID NO:136 (encoded by SEQ IDNO:135), can have a collagenase activity; a polypeptide having asequence as set forth in SEQ ID NO:142 (encoded by SEQ ID NO:142), canhave a neutral proteinase activity; a polypeptide having a sequence asset forth in SEQ ID NO:146 (encoded by SEQ ID NO:147), can have a serineprotease activity; a polypeptide having a sequence as set forth in SEQID NO:151 (encoded by SEQ ID NO:150), can have a metalloproteinaseactivity or an aspartyl proteinase (aspartyl protease) activity; apolypeptide having a sequence as set forth in SEQ ID NO:159 (encoded bySEQ ID NO:158), can have a metalloproteinase activity or ancarboxypeptidase activity (e.g., a serine-type carboxypeptidaseactivity); a sequence as set forth in SEQ ID NO:165 (encoded by SEQ IDNO:164), can have a peptidase activity, such as an aminopeptidaseactivity (e.g., a leucine aminopeptidase activity); a polypeptide havinga sequence as set forth in SEQ ID NO:172 (encoded by SEQ ID NO:171), canhave a peptidase or a CaaX prenyl protease activity (e.g., a CaaXprocessing activity); a polypeptide having a sequence as set forth inSEQ ID NO:180 (encoded by SEQ ID NO:179), can have a carboxypeptidaseactivity (e.g., a zinc carboxypeptidase activity); a polypeptide havinga sequence as set forth in SEQ ID NO:188 (encoded by SEQ ID NO:187), canhave a serine proteinase activity or a subtilase-like activity; apolypeptide having a sequence as set forth in SEQ ID NO:194 (encoded bySEQ ID NO:193), can have a metalloproteinase activity or a peptidaseactivity (e.g., an aminopeptidase activity); a polypeptide having asequence as set forth in SEQ ID NO:200 (encoded by SEQ ID NO:199), canhave a carboxypeptidase activity (e.g., a carboxypeptidase A activity ora zinc carboxypeptidase activity); a polypeptide having a sequence asset forth in SEQ ID NO:205 (encoded by SEQ ID NO:204), can have acarboxypeptidase activity (e.g., a zinc carboxypeptidase activity); apolypeptide having a sequence as set forth in SEQ ID NO:211 (encoded bySEQ ID NO:210), can have a carboxypeptidase activity (e.g., acarboxypeptidase S1 activity or a serine carboxypeptidase activity); apolypeptide having a sequence as set forth in SEQ ID NO:218 (encoded bySEQ ID NO:219), can have a zinc carboxypeptidase activity; a polypeptidehaving a sequence as set forth in SEQ ID NO:223 (encoded by SEQ IDNO:222), can have a peptidase activity; a polypeptide having a sequenceas set forth in SEQ ID NO:230 (encoded by SEQ ID NO:229), can have analkaline or serine proteinase activity or a subtilase activity; apolypeptide having a sequence as set forth in SEQ ID NO:235 (encoded bySEQ ID NO:234), can have a metalloproteinase activity or anacylaminoacyl peptidase activity (e.g., a carboxypeptidase S1 activity);a polypeptide having a sequence as set forth in SEQ ID NO:242 (encodedby SEQ ID NO:241), can have a carboxypeptidase activity (e.g., a zinccarboxypeptidase activity); a polypeptide having a sequence as set forthin SEQ ID NO:248 (encoded by SEQ ID NO:249), can have an aspartylprotease activity; a polypeptide having a sequence as set forth in SEQID NO:255 (encoded by SEQ ID NO:254), can have a metalloproteinaseactivity or an carboxypeptidase activity (e.g., a serine-typecarboxypeptidase activity). Any polypeptide of the invention, includingpolypeptides having the above-listed exemplary activities, may needprocessing (e.g., processing of a prepro form, phosphorylation,prenylation, dimerization, etc.) to generate the enzymatically activeform of the enzyme.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a protease activity which is thermostable. Thepolypeptide can retain a protease activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a protease activity which is thermotolerant. Thepolypeptide can retain a protease activity after exposure to atemperature in the range from greater than 37° C. to about 95° C. oranywhere in the range from greater than 55° C. to about 85° C. Thepolypeptide can retain a protease activity after exposure to atemperature in the range between about 1° C. to about 5° C., betweenabout 5° C. to about 15° C., between about 15° C. to about 25° C.,between about 25° C. to about 37° C., between about 37° C. to about 95°C., between about 55° C. to about 85° C., between about 70° C. to about75° C., or between about 90° C. to about 95° C., or more. In one aspect,the polypeptide retains a protease activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidcomprising a sequence as set forth in SEQ ID NO:1; SEQ ID NO:3; SEQ IDNO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ IDNO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ IDNO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ IDNO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ IDNO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ IDNO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ IDNO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ IDNO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ IDNO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ IDNO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ IDNO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123;SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ IDNO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158;SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ IDNO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248and/or SEQ ID NO:254, or fragments or subsequences thereof. In oneaspect, the nucleic acid encodes a polypeptide having a proteaseactivity. The nucleic acid can be at least about 10, 15, 20, 25, 30, 35,40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or moreresidues in length or the full length of the gene or transcript. In oneaspect, the stringent conditions include a wash step comprising a washin 0.2×SSC at a temperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a protease activity, wherein theprobe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more,consecutive bases of a sequence comprising a sequence of the invention,or fragments or subsequences thereof, wherein the probe identifies thenucleic acid by binding or hybridization. The probe can comprise anoligonucleotide comprising at least about 10 to 50, about 20 to 60,about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases ofa sequence comprising a sequence of the invention, or fragments orsubsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a protease activity, wherein theprobe comprises a nucleic acid comprising a sequence at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to a nucleic acid of theinvention, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence of the invention, or asubsequence thereof.

The invention provides an amplification primer pair for amplifying anucleic acid encoding a polypeptide having a protease activity, whereinthe primer pair is capable of amplifying a nucleic acid comprising asequence of the invention, or fragments or subsequences thereof. One oreach member of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 consecutive bases ofthe sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of the complementary strand ofthe first member.

The invention provides protease-encoding nucleic acids generated byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. The invention providesproteases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a protease by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having a protease activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a corn seed, a wheat kernel, an oilseed, arapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesameseed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a protease message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention. In one aspect, the antisenseoligonucleotide is between about 10 to 50, about 20 to 60, about 30 to70, about 40 to 80, or about 60 to 100 bases in length.

The invention provides methods of inhibiting the translation of aprotease message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesdouble-stranded inhibitory RNA (RNAi) molecules comprising a subsequenceof a sequence of the invention. In one aspect, the RNAi is about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.The invention provides methods of inhibiting the expression of aprotease in a cell comprising administering to the cell or expressing inthe cell a double-stranded inhibitory RNA (iRNA), wherein the RNAcomprises a subsequence of a sequence of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention over a region of atleast about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350 or more residues, or over the full length of the polypeptide,and the sequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection. Exemplary polypeptide orpeptide sequences of the invention include SEQ ID NO:2; SEQ ID NO:4; SEQID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ IDNO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ IDNO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ IDNO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ IDNO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ IDNO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ IDNO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ IDNO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ IDNO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ IDNO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ IDNO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124;SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ IDNO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165;SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ IDNO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255,or a protease encoded by SEQ ID NO:145, and subsequences thereof andvariants thereof. Exemplary polypeptides also include fragments of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600 or more residues in length, or overthe full length of an enzyme. Exemplary polypeptide or peptide sequencesof the invention include sequence encoded by a nucleic acid of theinvention. Exemplary polypeptide or peptide sequences of the inventioninclude polypeptides or peptides specifically bound by an antibody ofthe invention. In one aspect, a polypeptide of the invention can have atleast one protease activity.

Another aspect of the invention provides an isolated or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutivebases of a polypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto. The peptide can be, e.g., an immunogenic fragment, a motif(e.g., a binding site), a signal sequence (e.g., as in Table 4), aprepro sequence or an active site.

In one aspect, protease activity comprises catalyzing hydrolysis ofpeptide bonds. The protease activity can comprise an endoproteaseactivity and/or an exoprotease activity. The protease activity cancomprise a carboxypeptidase activity, an aminopeptidase activity, aserine protease activity, a metalloprotease activity, a cysteineprotease activity and/or an aspartic protease activity. In one aspect,protease activity can comprise activity the same or similar to achymotrypsin, a trypsin, an elastase, a kallikrein and/or a subtilisinactivity. The protease activity can comprise a peptidase activity, suchas a dipeptidylpeptidase or a carboxypeptidase activity.

In one aspect, the protease activity is thermostable. The polypeptidecan retain a protease activity under conditions comprising a temperaturerange of between about 1° C. to about 5° C., between about 5° C. toabout 15° C., between about 15° C. to about 25° C., between about 25° C.to about 37° C., between about 37° C. to about 95° C., between about 55°C. to about 85° C., between about 70° C. to about 75° C., or betweenabout 90° C. to about 95° C., or more. In another aspect, the proteaseactivity can be thermotolerant. The polypeptide can retain a proteaseactivity after exposure to a temperature in the range from greater than37° C. to about 95° C., or in the range from greater than 55° C. toabout 85° C. In one aspect, the polypeptide can retain a proteaseactivity after exposure to a temperature in the range from greater than90° C. to about 95° C. at pH 4.5.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous protease or non-protease signal sequence.

In one aspect, the invention provides a signal sequence comprising apeptide comprising/consisting of a sequence as set forth in residues 1to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1to 44 (or a longer peptide) of a polypeptide of the invention. In oneaspect, the invention provides a signal sequence comprising a peptidecomprising/consisting of a sequence as set forth Table 4.

The invention provides isolated or recombinant peptides comprising anamino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to residues 1 to 37 of SEQ ID NO:2, at least 95%, 96%,97%, 98%, 99%, or more sequence identity to residues 1 to 36 of SEQ IDNO:4, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity toresidues 1 to 32 of SEQ ID NO:6, at least 95%, 96%, 97%, 98%, 99%, ormore sequence identity to residues 1 to 28 of SEQ ID NO:10, at least95%, 96%, 97%, 98%, 99%, or more sequence identity to residues 1 to 33of SEQ ID NO:14, and least 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to the other signal sequences as set forth in the SEQ IDlisting, wherein the sequence identities are determined by analysis witha sequence comparison algorithm or by visual inspection. These peptidescan act as signal sequences on its endogenous protease, on anotherprotease, or a heterologous protein (a non-protease enzyme or otherprotein).

In one aspect, the invention provides chimeric proteins comprising afirst domain comprising a signal sequence of the invention (e.g., seeTable 4) and at least a second domain. The protein can be a fusionprotein. The second domain can comprise an enzyme. The enzyme can be aprotease.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) of the invention and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro sequence and/or catalytic domain (CD). Inone aspect, the heterologous polypeptide or peptide is not a protease.The heterologous polypeptide or peptide can be amino terminal to,carboxy terminal to or on both ends of the signal peptide (SP), preprosequence and/or catalytic domain (CD).

The invention provides isolated or recombinant nucleic acids encoding achimeric polypeptide, wherein the chimeric polypeptide comprises atleast a first domain comprising signal peptide (SP), a prepro domainand/or a catalytic domain (CD) of the invention and at least a seconddomain comprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro domain and/or catalytic domain (CD).

In one aspect, the protease activity comprises a specific activity atabout 37° C. in the range from about 1 to about 1200 units per milligramof protein, or, about 100 to about 1000 units per milligram of protein.In another aspect, the protease activity comprises a specific activityfrom about 100 to about 1000 units per milligram of protein, or, fromabout 500 to about 750 units per milligram of protein. Alternatively,the protease activity comprises a specific activity at 37° C. in therange from about 1 to about 750 units per milligram of protein, or, fromabout 500 to about 1200 units per milligram of protein. In one aspect,the protease activity comprises a specific activity at 37° C. in therange from about 1 to about 500 units per milligram of protein, or, fromabout 750 to about 1000 units per milligram of protein. In anotheraspect, the protease activity comprises a specific activity at 37° C. inthe range from about 1 to about 250 units per milligram of protein.Alternatively, the protease activity comprises a specific activity at37° C. in the range from about 1 to about 100 units per milligram ofprotein. In another aspect, the thermotolerance comprises retention ofat least half of the specific activity of the protease at 37° C. afterbeing heated to the elevated temperature. Alternatively, thethermotolerance can comprise retention of specific activity at 37° C. inthe range from about 1 to about 1200 units per milligram of protein, or,from about 500 to about 1000 units per milligram of protein, after beingheated to the elevated temperature. In another aspect, thethermotolerance can comprise retention of specific activity at 37° C. inthe range from about 1 to about 500 units per milligram of protein afterbeing heated to the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain a protease activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain a protease activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 11. In one aspect, the polypeptide can retain aprotease activity after exposure to conditions comprising about pH 6.5,pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptidecan retain a protease activity after exposure to conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH11.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different protease, a different enzyme or anotherprotein. In one aspect, the second domain can be a polypeptide and theheterodimer can be a fusion protein. In one aspect, the second domaincan be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having a proteaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides food supplements for an animal comprising apolypeptide of the invention, e.g., a polypeptide encoded by the nucleicacid of the invention. In one aspect, the polypeptide in the foodsupplement can be glycosylated. The invention provides edible enzymedelivery matrices comprising a polypeptide of the invention, e.g., apolypeptide encoded by the nucleic acid of the invention. In one aspect,the delivery matrix comprises a pellet. In one aspect, the polypeptidecan be glycosylated. In one aspect, the protease activity isthermotolerant. In another aspect, the protease activity isthermostable.

The invention provides method of isolating or identifying a polypeptidehaving a protease activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypcptide, thereby isolating or identifying apolypeptide having a protease activity.

The invention provides methods of making an anti-protease antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-protease antibody. The invention provides methods ofmaking an anti-protease immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having aprotease activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing a protease substrate; and (c) contactingthe polypeptide or a fragment or variant thereof of step (a) with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having a protease activity.

The invention provides methods for identifying a protease substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as a protease substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of a proteaseactivity comprising the following steps: (a) providing a polypeptide ofthe invention or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a test compound; (c) contacting the polypeptideof step (a) with the test compound of step (b) and measuring an activityof the protease, wherein a change in the protease activity measured inthe presence of the test compound compared to the activity in theabsence of the test compound provides a determination that the testcompound modulates the protease activity. In one aspect, the proteaseactivity can be measured by providing a protease substrate and detectinga decrease in the amount of the substrate or an increase in the amountof a reaction product, or, an increase in the amount of the substrate ora decrease in the amount of a reaction product. A decrease in the amountof the substrate or an increase in the amount of the reaction productwith the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an activator of protease activity. An increase in the amount of thesubstrate or a decrease in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an inhibitorof protease activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a protease activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having a protease activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having aprotease activity from an environmental sample. One or each member ofthe amplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence ofthe invention. In one aspect, the amplification primer sequence pair isan amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a protease activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof; (b) isolating a nucleic acid from the environmentalsample or treating the environmental sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treatedenvironmental sample of step (b) with the polynucleotide probe of step(a); and (d) isolating a nucleic acid that specifically hybridizes withthe polynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide having a protease activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having a protease activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant protease polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until a proteasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant protease polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant proteasepolypeptide has increased glycosylation as compared to the proteaseencoded by a template nucleic acid. Alternatively, the variant proteasepolypeptide has a protease activity under a high temperature, whereinthe protease encoded by the template nucleic acid is not active underthe high temperature. In one aspect, the method can be iterativelyrepeated until a protease coding sequence having an altered codon usagefrom that of the template nucleic acid is produced. In another aspect,the method can be iteratively repeated until a protease gene havinghigher or lower level of message expression or stability from that ofthe template nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a protease activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving a protease activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a protease activity; the method comprisingthe following steps: (a) providing a nucleic acid of the invention; and,(b) identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding a protease.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a protease activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a proteasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having a protease activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified protease active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes a protease active site or aprotease substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified protease active sites or substrate binding sites.In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) by a method comprising an optimized directed evolutionsystem, gene site-saturation mutagenesis (GSSM), synthetic ligationreassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)and a combination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises a protease enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Theinvention provides methods for modifying a small molecule comprising thefollowing steps: (a) providing a protease enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the protease enzyme, thereby modifying a smallmolecule by a protease enzymatic reaction. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by the protease enzyme. Inone aspect, the method can comprise a plurality of additional enzymesunder conditions that facilitate a plurality of biocatalytic reactionsby the enzymes to form a library of modified small molecules produced bythe plurality of enzymatic reactions. In another aspect, the method canfurther comprise the step of testing the library to determine if aparticular modified small molecule which exhibits a desired activity ispresent within the library. The step of testing the library can furthercomprise the steps of systematically eliminating all but one of thebiocatalytic reactions used to produce a portion of the plurality of themodified small molecules within the library by testing the portion ofthe modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofa protease enzyme comprising the steps of: (a) providing a proteaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof; and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence fora protease activity, thereby detemining a functional fragment of aprotease enzyme. In one aspect, the protease activity is measured byproviding a protease substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of a protease polypeptide, the method comprisingglycosylating a protease polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe protease polypeptide. In one aspect, the protease specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant proteasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell. The invention provides methods of expressing aheterologous nucleic acid sequence in a plant cell comprising thefollowing steps: (a) transforming the plant cell with a heterologousnucleic acid sequence operably linked to a promoter, wherein theheterologous nucleic sequence comprises a sequence of the invention; (b)growing the plant under conditions wherein the heterologous nucleicacids sequence is expressed in the plant cell.

The invention provides methods for hydrolyzing, breaking up ordisrupting a protein-comprising composition comprising the followingsteps: (a) providing a polypeptide of the invention having a proteaseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing a composition comprising a protein; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the protease hydrolyzes, breaks up or disrupts theprotein-comprising composition. In one aspect, the composition comprisesa plant cell, a bacterial cell, a yeast cell, an insect cell, or ananimal cell. Thus, the composition can comprise any plant or plant part,any protein-containing food or feed, a waste product and the like. Theinvention provides methods for liquefying or removing a protein from acomposition comprising the following steps: (a) providing a polypeptideof the invention having a protease activity, or a polypeptide encoded bya nucleic acid of the invention; (b) providing a composition comprisinga protein; and (c) contacting the polypeptide of step (a) with thecomposition of step (b) under conditions wherein the protease removes orliquefies the protein.

The invention provides detergent compositions comprising a polypeptideof the invention, or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide has a protease activity. The proteasecan be a nonsurface-active protease or a surface-active protease. Theprotease can be formulated in a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a gelform, a paste or a slurry form. The invention provides methods forwashing an object comprising the following steps: (a) providing acomposition comprising a polypeptide of the invention having a proteaseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing an object; and (c) contacting the polypeptide of step (a)and the object of step (b) under conditions wherein the composition canwash the object.

The invention provides textiles or fabrics, including, e.g., threads,comprising a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention. In one aspect, the textiles or fabricscomprise cellulose-containing fibers. The invention provides methods forremoving protein stains from a composition comprising the followingsteps: (a) providing a composition comprising a polypeptide of theinvention having a protease activity, or a polypeptide encoded by anucleic acid of the invention; (b) providing a composition having aprotein stain; and (c) contacting the polypeptide of step (a) and thecomposition of step (b) under conditions wherein the protease can removethe stain. The invention provides methods for improving the finish of afabric comprising the following steps: (a) providing a compositioncomprising a polypeptide of the invention having a protease activity, ora polypeptide encoded by a nucleic acid of the invention; (b) providinga fabric; and (c) contacting the polypeptide of step (a) and the fabricof step (b) under conditions wherein the polypeptide can treat thefabric thereby improving the finish of the fabric. In one aspect, thefabric is a wool or a silk.

The invention provides feeds or foods comprising a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention.The invention provides methods for hydrolyzing proteins in a feed or afood prior to consumption by an animal comprising the following steps:(a) obtaining a feed material comprising a protease of the invention, ora protease encoded by a nucleic acid of the invention; and (b) addingthe polypeptide of step (a) to the feed or food material in an amountsufficient for a sufficient time period to cause hydrolysis of theprotein and formation of a treated food or feed, thereby hydrolyzing theproteins in the food or the feed prior to consumption by the animal. Inone aspect, the invention provides methods for hydrolyzing proteins in afeed or a food after consumption by an animal comprising the followingsteps: (a) obtaining a feed material comprising a protease of theinvention, or a protease encoded by a nucleic acid of the invention; (b)adding the polypeptide of step (a) to the feed or food material; and (c)administering the feed or food material to the animal, wherein afterconsumption, the protease causes hydrolysis of the proteins in the feedor food in the digestive tract of the animal. The food or the feed canbe, e.g., corn.

The invention provides methods for improving texture and flavor of adairy product comprising the following steps: (a) providing apolypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention; (b) providing a dairyproduct; and (c) contacting the polypeptide of step (a) and the dairyproduct of step (b) under conditions wherein the protease can improvethe texture or flavor of the dairy product. In one aspect, the dairyproduct comprises a cheese or a yogurt. The invention provides dairyproducts comprising a protease of the invention, or is encoded by anucleic acid of the invention. The invention provides methods fortenderizing a meat or a fish comprising the following steps: (a)providing a polypeptide of the invention having a protease activity, ora protease encoded by a nucleic acid of the invention; (b) providing acomposition comprising meat or fish; and (c) contacting the polypeptideof step (a) and the composition of step (b) under conditions wherein thepolypeptide can tenderize the meat or the fish. The invention providesmethods for producing a gluten-free product comprising the followingsteps: (a) providing a polypeptide of the invention having a proteaseactivity, or a protease encoded by a nucleic acid of the invention; (b)providing a product comprising gluten; and (c) contacting thepolypeptide of step (a) and the product of step (b) under conditionswherein the polypeptide can hydrolyze gluten thereby producing thegluten-free product. In one aspect, the gluten-free product is a cereal,a bread or a beer. The invention provides gluten-free food compositionscomprising a polypeptide of the invention, or a protease encoded by anucleic acid of the invention, wherein the polypeptide comprises aprotease activity.

The invention provides methods for improving the extraction of oil froman oil-rich plant material comprising the following steps: (a) providinga polypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention; (b) providing an oil-richplant material; and (c) contacting the polypeptide of step (a) and theoil-rich plant material. In one aspect, the oil-rich plant materialcomprises an oil-rich seed. The oil can be a soybean oil, an olive oil,a rapeseed (canola) oil or a sunflower oil.

The invention provides methods for preparing a fruit or vegetable juice,syrup, puree or extract comprising the following steps: (a) providing apolypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention; (b) providing a compositionor a liquid comprising a fruit or vegetable material; and (c) contactingthe polypeptide of step (a) and the composition, thereby preparing thefruit or vegetable juice, syrup, puree or extract.

The invention provides papers or paper products or paper pulp comprisinga protease of the invention, or a polypeptide encoded by a nucleic acidof the invention. The invention provides methods for treating a paper ora paper or wood pulp comprising the following steps: (a) providing apolypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention; (b) providing a compositioncomprising a paper or a paper or wood pulp; and (c) contacting thepolypeptide of step (a) and the composition of step (b) under conditionswherein the protease can treat the paper or paper or wood pulp.

The invention provides pharmaceutical compositions comprising apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention. In one aspect, the pharmaceutical composition acts asa digestive aid or as a topical skin care. The invention providesmethods of treating an imbalance of desquamation comprising topicalapplication of a pharmaceutical composition of the invention. In oneaspect, the treatment is prophylactic. The invention provides oral careproducts comprising a polypeptide of the invention having a proteaseactivity, or a protease encoded by a nucleic acid of the invention. Theoral care product can comprise a toothpaste, a dental cream, a gel or atooth powder, an odontic, a mouth wash, a pre- or post brushing rinseformulation, a chewing gum, a lozenge or a candy. The invention providescontact lens cleaning compositions comprising a polypeptide of theinvention having a protease activity, or a protease encoded by a nucleicacid of the invention.

The invention provides methods for treating solid or liquid animal wasteproducts comprising the following steps: (a) providing a polypeptide ofthe invention having a protease activity, or a protease encoded by anucleic acid of the invention; (b) providing a solid or a liquid animalwaste; and (c) contacting the polypeptide of step (a) and the solid orliquid waste of step (b) under conditions wherein the protease can treatthe waste. The invention provides processed waste products comprising apolypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention.

The invention provides hairball prevention and/or remedies comprising apolypeptide of the invention having a protease activity, or a proteaseencoded by a nucleic acid of the invention. The invention provides bloodor organic spot removers comprising a polypeptide of the inventionhaving a protease activity, or a protease encoded by a nucleic acid ofthe invention.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 is a an illustration of results of testing SEQ ID NO:144 (encodedby SEQ ID NO:143) in a gelatin in fluorescent liquid end point assay, asdescribed in detail in Example 1, below.

FIG. 6 is an illustration of a standard curve of (pNA)(para-nitroanalide), generated to allow conversion of pNA absorbance(A405 nm) to moles of pNA, as described in detail in Example 1, below.

FIG. 7 is an illustration of a standard curve of subtilisin A protease,as described in detail in Example 1, below.

FIG. 8 is a an illustration of results if a protease activity using thesmall peptide substrate p-nitroanalide linkedAlanine-Alanine-Proline-Phenylalanine, as described in detail in Example1, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides polypeptides having a protease activity,polynucleotides encoding the polypeptides, and methods for making andusing these polynucleotides and polypeptides. In one aspect, theproteases of the invention are used to catalyze the hydrolysis ofpeptide bonds. The proteases of the invention can be used to make and/orprocess foods or feeds, textiles, detergents and the like. The proteasesof the invention can be used in pharmaceutical compositions and dietaryaids.

The protease preparations of the invention (including those for treatingor processing feeds or foods, treating fibers and textiles, wastetreatments, plant treatments, and the like) can further comprise one ormore enzymes, for example, pectate lyases, cellulases(endo-beta-1,4-glucanases), beta-glucanases(endo-beta-1,3(4)-glucanases), lipases, cutinases, peroxidases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases, transglutaminases; or mixturesthereof.

Definitions

The term “protease” includes all polypeptides having a proteaseactivity, including a peptidase and/or a proteinase activity. A proteaseactivity of the invention can comprise catalysis of the hydrolysis ofpeptide bonds. The proteases of the invention can catalyze peptidehydrolysis reactions in both directions. The direction of the reactioncan be determined, e.g., by manipulating substrate and/or productconcentrations, temperature, selection of protease and the like. Theprotease activity can comprise an endoprotease activity and/or anexoprotease activity. The protease activity can comprise a proteaseactivity, e.g., a carboxypeptidase activity, a dipeptidylpeptidase or anaminopeptidase activity, a serine protease activity, a metalloproteinaseactivity, a cysteine protease activity and/or an aspartic proteaseactivity. In one aspect, protease activity can comprise activity thesame or similar to a chymotrypsin, a trypsin, an elastase, a kallikreinand/or a subtilisin activity.

In describing a polypeptide of the invention having a protease activity,e.g., an exemplary polypeptide having a sequence as set forth in SEQ IDNO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12;SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22;SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32;SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42;SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:52;SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62;SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72;SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82;SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92;SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102;SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ IDNO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130;SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ IDNO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188;SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ IDNO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQID NO:249; SEQ ID NO:255; a polypeptide encoded by SEQ ID NO:145, it ismeant that the polypeptide has a protease activity with and/or without asignal sequence, or, with and/or without a prepro sequence (e.g., a“prepro” domain), if the polypeptide has a signal sequence and/or aprepro sequence (e.g., a “prepro” domain). Thus, the invention includespolypeptides (having a protease activity) in inactive form, e.g., as aproprotein before “maturation” or processing of its prepro sequence(e.g., by a proprotein-processing enzyme, such as a proproteinconvertase) to generate an “active” mature protein, or, before“activation” by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike, in addition to including all active forms and active subsequences(e.g., catalytic domains or active sites) of the protease.

A polypeptide can be routinely assayed for protease activity (e.g.,tested to see if the protein is within the scope of the invention) byany method, e.g., protease activity can be assayed by the hydrolysis ofcasein in zymograms, the release of fluorescence from gelatin, or therelease of p-nitroanalide from various small peptide substrates (theseand other exemplary protease assays are set forth in the Examples,below).

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a protease of the invention)in a host compatible with such sequences. Expression cassettes includeat least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers. Thus, expressioncassettes also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatorysequence to a transcribed sequence. For example, a promoter is operablylinked to a coding sequence, such as a nucleic acid of the invention, ifit stimulates or modulates the transcription of the coding sequence inan appropriate host cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan

The term “gene” includes a nucleic acid sequence comprising a segment ofDNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include regionspreceding and following the coding region, such as leader and trailer,promoters and enhancers, as well as, where applicable, interveningsequences (introns) between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. The term also includesglycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

The term “isolated” includes a material removed from its originalenvironment, e.g., the natural environment if it is naturally occurring.For example, a naturally occurring polynucleotide or polypeptide presentin a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition. Individual nucleic acids obtained from a library can beconventionally purified to electrophoretic homogeneity. In alternativeaspects, the invention provides nucleic acids which have been purifiedfrom genomic DNA or from other sequences in a library or otherenvironment by at least one, two, three, four, five or more orders ofmagnitude.

As used herein, the term “recombinant” can include nucleic acidsadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. In one aspect, nucleic acids represent 5% or moreof the number of nucleic acid inserts in a population of nucleic acid“backbone molecules.” “Backbone molecules” according to the inventioninclude nucleic acids such as expression vectors, self-replicatingnucleic acids, viruses, integrating nucleic acids, and other vectors ornucleic acids used to maintain or manipulate a nucleic acid insert ofinterest. In one aspect, the enriched nucleic acids represent 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the numberof nucleic acid inserts in the population of recombinant backbonemolecules. “Recombinant” polypeptides or proteins refer to polypeptidesor proteins produced by recombinant DNA techniques; e.g., produced fromcells transformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence can be “operably linked to” a coding sequence whenRNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” includes either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, can refer to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one any known sequencecomparison algorithm, as discussed in detail below, or by visualinspection. In alternative aspects, the invention provides nucleic acidand polypeptide sequences having substantial identity to an exemplarysequence of the invention, e.g., SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5;SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ IDNO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ IDNO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ IDNO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ IDNO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ IDNO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ IDNO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ IDNO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ IDNO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ IDNO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125;SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ IDNO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164;SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ IDNO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 and/or SEQ IDNO:254 (nucleic acids); SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ IDNO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ IDNO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ IDNO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ IDNO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ IDNO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ IDNO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ IDNO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ IDNO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ IDNO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ IDNO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116;SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ IDNO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144;SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ IDNO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230;SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or thepolypeptide encoded by SEQ ID NO:145, over a region of at least about10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues, or aregion ranging from between about 50 residues to the full length of thenucleic acid or polypeptide. Nucleic acid sequences of the invention canbe substantially identical over the entire length of a polypeptidecoding region.

A “substantially identical” amino acid sequence also can include asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from a protease, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for protease activity can be removed.

“Hybridization” includes the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Stringent conditions can be defined by, for example,the concentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. For example, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature, altering the timeof hybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

“Variant” includes polynucleotides or polypeptides of the inventionmodified at one or more base pairs, codons, introns, exons, or aminoacid residues (respectively) yet still retain the biological activity ofa protease of the invention (which can be assayed by, e.g., thehydrolysis of casein in zymograms, the release of fluorescence fromgelatin, or the release of p-nitroanalide from various small peptidesubstrates). Variants can be produced by any number of means includedmethods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM and any combination thereof.Techniques for producing variant protease having activity at a pH ortemperature, for example, that is different from a wild-type protease,are included herein.

The term “saturation mutagenesis” or “GSSM” includes a method that usesdegenerate oligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids (e.g., SEQ ID NO:1; SEQ ID NO:3;SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ IDNO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ IDNO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ IDNO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ IDNO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ IDNO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ IDNO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ IDNO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ IDNO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ IDNO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:11; SEQ ID NO:113; SEQID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123;SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ IDNO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158;SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ IDNO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248and/or SEQ ID NO:254; nucleic acids encoding polypeptides as set forthin SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ IDNO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ IDNO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ IDNO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ IDNO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ IDNO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ IDNO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ IDNO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ IDNO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ IDNO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120;SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ IDNO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151;SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ IDNO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242;SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ IDNO:145, including expression cassettes such as expression vectors,encoding the polypeptides of the invention. The invention also includesmethods for discovering new protease sequences using the nucleic acidsof the invention. The invention also includes methods for inhibiting theexpression of protease genes, transcripts and polypeptides using thenucleic acids of the invention. Also provided are methods for modifyingthe nucleic acids of the invention by, e.g., synthetic ligationreassembly, optimized directed evolution system and/or saturationmutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., proteases) generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lacI promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter.

Other promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express a protease of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express a protease of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol. 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassaya vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofprotease-expressing nucleic acid in a specific tissue, organ or celltype (i.e. tissue-specific promoters) or may be otherwise under moreprecise environmental or developmental control or under the control ofan inducible promoter. Examples of environmental conditions that mayaffect transcription include anaerobic conditions, elevated temperature,the presence of light, or sprayed with chemicals/hormones. For example,the invention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically—(e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the protease-producing nucleic acids of the invention willallow the grower to select plants with the optimal protease expressionand/or activity. The development of plant parts can thus controlled. Inthis way the invention provides the means to facilitate the harvestingof plants and plant parts. For example, in various embodiments, themaize In2-2 promoter, activated by benzenesulfonamide herbicidesafeners, is used (De Veylder (1997) Plant Cell Physiol. 38:568-577);application of different herbicide safeners induces distinct geneexpression patterns, including expression in the root, hydathodes, andthe shoot apical meristem. Coding sequences of the invention are alsounder the control of a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theproteases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements. In one aspect,the expression vectors contain one or more selectable marker genes topermit selection of host cells containing the vector. Such selectablemarkers include genes encoding dihydrofolate reductase or genesconferring neomycin resistance for eukaryotic cell culture, genesconferring tetracycline or ampicillin resistance in E. coli, and the S.cerevisiae TRP1 gene. Promoter regions can be selected from any desiredgene using chloramphenicol transferase (CAT) vectors or other vectorswith selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp.; potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a protease of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477; U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are preferred.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the proteases of the invention, or modified nucleic acidsof the invention, can be reproduced by amplification. Amplification canalso be used to clone or modify the nucleic acids of the invention.Thus, the invention provides amplification primer sequence pairs foramplifying nucleic acids of the invention. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 or more residues of a nucleic acid of theinvention, and about the first (the 5′) 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 residues of the complementary strand (e.g., of SEQ IDNO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11;SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21;SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31;SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41;SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51;SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61;SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71;SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81;SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91;SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101;SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ IDNO:11; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129;SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ IDNO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179;SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ IDNO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQID NO:241; SEQ ID NO:248 and/or SEQ ID NO:254).

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a proteaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesproteases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a protease by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(e.g., SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9;SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19;SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29;SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39;SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49;SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59;SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69;SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79;SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89;SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99;SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; SEQ IDNO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117; SEQID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127;SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQ IDNO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ ID NO:171;SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQ IDNO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229; SEQID NO:234; SEQ ID NO:241; SEQ ID NO:248 and/or SEQ ID NO:254, andnucleic acids encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144and/or SEQ ID NO:147) over a region of at least about 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550 or more, residues. The invention provides polypeptidescomprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to an exemplarypolypeptide of the invention. The extent of sequence identity (homology)may be determined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W.H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence, e.g., asequence of the invention, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the numbers of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary polypeptide ornucleic acid sequence of the invention are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary polypeptide or nucleic acidsequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a sequence of the invention, that sequence iswithin the scope of the invention. In alternative embodiments,subsequences ranging from about 20 to 600, about 50 to 200, and about100 to 150 are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequence for comparison are well known in theart. Optimal aligmnent of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482, 1981, by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity methodof person & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection. Other algorithms for determining homology or identityinclude, for example, in addition to a BLAST program (Basic LocalAlignment Search Tool at the National Center for BiologicalInformation), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS(Protein Multiple Sequence Alignment), ASSET (Aligned SegmentStatistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (BiologicalSequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher),FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS,LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegasalgorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used, default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “−F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the inventioninclude:

“Filter for low complexity: ON

Word Size: 3

Matrix: Blosum62

Gap Costs: Existence:11

Extension: 1”

Other default settings can be: filter for low complexity OFF, word sizeof 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and agap extension penalty of −1. An exemplary NCBI BLAST 2.2.2 programsetting has the “−W” option default to 0. This means that, if not set,the word size defaults to 3 for proteins and 1 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. Accordingly, the invention provides computers,computer systems, computer readable mediums, computer programs productsand the like recorded or stored thereon the nucleic acid and polypeptidesequences of the invention. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any known methods forrecording information on a computer readable medium to generatemanufactures comprising one or more of the nucleic acid and/orpolypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data.

The processor 105 can be any well-known type of central processing unit,such as, for example, the Pentium III from Intel Corporation, or similarprocessor from Sun, Motorola, Compaq, AMD or International BusinessMachines. The computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data, and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110. The data retrieving device 118 mayrepresent, for example, a floppy disk drive, a compact disk drive, amagnetic tape drive, or a modem capable of connection to a remote datastorage system (e.g., via the internet) etc. In some embodiments, theinternal data storage device 110 is a removable computer readable mediumsuch as a floppy disk, a compact disk, a magnetic tape, etc. containingcontrol logic and/or data recorded thereon. The computer system 100 mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. The computersystem 100 includes a display 120 which is used to display output to acomputer user. It should also be noted that the computer system 100 canbe linked to other computer systems 125 a-c in a network or wide areanetwork to provide centralized access to the computer system 100.Software for accessing and processing the nucleotide or amino acidsequences of the invention can reside in main memory 115 duringexecution. In some aspects, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence ofthe invention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of the invention stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device. The process 200 then moves to a state 204wherein a database of sequences is opened for analysis and comparison.The process 200 then moves to a state 206 wherein the first sequencestored in the database is read into a memory on the computer. Acomparison is then performed at a state 210 to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database. It should benoted that if a determination had been made at the decision state 212that the sequences were not homologous, then the process 200 would moveimmediately to the decision state 218 in order to determine if any othersequences were available in the database for comparison. Accordingly,one aspect of the invention is a computer system comprising a processor,a data storage device having stored thereon a nucleic acid sequence ofthe invention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs, or it may identify structuralmotifs in sequences which are compared to these nucleic acid codes andpolypeptide codes. FIG. 3 is a flow diagram illustrating one embodimentof a process 250 in a computer for determining whether two sequences arehomologous. The process 250 begins at a start state 252 and then movesto a state 254 wherein a first sequence to be compared is stored to amemory. The second sequence to be compared is then stored to a memory ata state 256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with an every character in a secondsequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention can be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention (e.g., SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ IDNO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ IDNO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ IDNO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ IDNO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ IDNO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ IDNO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ IDNO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ IDNO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ IDNO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; SEQID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117;SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ IDNO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145;SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ IDNO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229;SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 and/or SEQ ID NO:254), or anucleic acid that encodes a polypeptide of the invention (e.g., SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ IDNO:140; SEQ ID NO:142; SEQ ID NO:144 and/or SEQ ID NO:147). Thestringent conditions can be highly stringent conditions, mediumstringent conditions and/or low stringent conditions, including the highand reduced stringency conditions described herein. In one aspect, it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention, as discussed below.

In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than fill length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na⁺ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization 10conditions is when the above hybridization is conducted at 30%formamide. A specific example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 10%formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with a proteaseactivity or fragments thereof or for identifying protease genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutivebases of a sequence as set forth in a nucleic acid of the invention. Theprobes identify a nucleic acid by binding and/or hybridization. Theprobes can be used in arrays of the invention, see discussion below,including, e.g., capillary arrays. The probes of the invention can alsobe used to isolate other nucleic acids or polypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequencespresent in the sample. Where necessary, conditions which permit theprobe to specifically hybridize to complementary sequences may bedetermined by placing the probe in contact with complementary sequencesfrom samples known to contain the complementary sequence, as well ascontrol sequences which do not contain the complementary sequence.Hybridization conditions, such as the salt concentration of thehybridization buffer, the formamide concentration of the hybridizationbuffer, or the hybridization temperature, may be varied to identifyconditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency can vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization can be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO4, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10× Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/μg) of ³²Pend-labeled oligonucleotide probe can then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is thelength of the probe. Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon spermDNA or 6×SSC, 5× Denhardt's reagent, 0.5% SDS, 100 μg denaturedfragmented salmon sperm DNA, 50% formamide. Formulas for SSC andDenhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na⁺ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 99%, 98%, 97%, at least 95%,at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55%, or at least 50% homology to anucleic acid sequence of the invention comprising at least about 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive basesthereof, and the sequences complementary thereto. Homology may bemeasured using an alignment algorithm, as discussed herein. For example,the homologous polynucleotides may have a coding sequence which is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variants may have a substitution,deletion or addition of one or more nucleotides when compared to anucleic acid of the invention.

Additionally, the probes and methods of the invention can be used toisolate nucleic acids which encode polypeptides having at least about99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least50% sequence identity (homology) to a polypeptide of the inventioncomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids, as determined using a sequence alignmentalgorithm (e.g., such as the FASTA version 3.0t78 algorithm with thedefault parameters, or a BLAST 2.2.2 program with exemplary settings asset forth herein).

Inhibiting Expression of Protease

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g.,protease-encoding nucleic acids. Antisense sequences are capable ofinhibiting the transport, splicing or transcription of protease-encodinggenes. The inhibition can be effected through the targeting of genomicDNA or messenger RNA. The transcription or function of targeted nucleicacid can be inhibited, for example, by hybridization and/or cleavage.One particularly useful set of inhibitors provided by the presentinvention includes oligonucleotides which are able to either bindprotease gene or message, in either case preventing or inhibiting theproduction or function of protease. The association can be throughsequence specific hybridization. Another useful class of inhibitorsincludes oligonucleotides which cause inactivation or cleavage ofprotease message. The oligonucleotide can have enzyme activity whichcauses such cleavage, such as ribozymes. The oligonucleotide can bechemically modified or conjugated to an enzyme or composition capable ofcleaving the complementary nucleic acid. A pool of many different sucholigonucleotides can be screened for those with the desired activity.Thus, the invention provides various compositions for the inhibition ofprotease expression on a nucleic acid and/or protein level, e.g.,antisense, iRNA and ribozymes comprising protease sequences of theinvention and the anti-protease antibodies of the invention.

Inhibition of protease expression can have a variety of industrialapplications. For example, inhibition of protease expression can slow orprevent spoilage. Spoilage can occur when polypeptides, e.g., structuralpolypeptides, are enzymatically degraded. This can lead to thedeterioration, or rot, of fruits and vegetables. In one aspect, use ofcompositions of the invention that inhibit the expression and/oractivity of proteases, e.g., antibodies, antisense oligonucleotides,ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in oneaspect, the invention provides methods and compositions comprisingapplication onto a plant or plant product (e.g., a fruit, seed, root,leaf, etc.) antibodies, antisense oligonucleotides, ribozymes and RNAiof the invention to slow or prevent spoilage. These compositions alsocan be expressed by the plant (e.g., a transgenic plant) or anotherorganism (e.g., a bacterium or other microorganism transformed with aprotease gene of the invention). The compositions of the invention forthe inhibition of protease expression (e.g., antisense, iRNA, ribozymes,antibodies) can be used as pharmaceutical compositions, e.g., asanti-pathogen agents or in other therapies, e.g., anti-inflammatory orskin or digestive aid treatments. For example, proteases are attractiveantimalarial targets because of their indispensable roles in parasiteinfection and development, especially in the processes of hosterythrocyte rupture, invasion and hemoglobin degradation; see, e.g., Wu(2003) Genome Res. 13:601-616. Selective inhibition of the mosquitoangiotensin-converting enzyme (ACE) (a dipeptidyl carboxypeptidase)involved in the activation/inactivation of a peptide regulatingegg-laying activity can be an effective anti-mosquito method; see, e.g.,Ekbote (2003) Comp. Biochem. Physiol. B. Biochem. Mol. Biol.134:593-598. Inhibition of matrix metalloproteases (e.g.,metalloproteinases) and collagenases, which can degrade extracellularmatrices and promote cancer cell migration and metastases, can be usedto treat or ameliorate these conditions; see e.g., Elnemr (2003) GastricCancer 6:30-38.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingprotease message which can inhibit proteolytic activity by targetingmRNA. Strategies for designing antisense oligonucleotides are welldescribed in the scientific and patent literature, and the skilledartisan can design such protease oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisenseprotease sequences of the invention (see, e.g., Gold (1995) J. of Biol.Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding protease message.These ribozymes can inhibit protease activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the protease-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a protease sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a protease gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a protease. These methodscan be repeated or used in various combinations to generate proteaseshaving an altered or different activity or an altered or differentstability from that of a protease encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’ ” Journal of Molecular Biology 255:373-386; Stemmer (1996)“Sexual PCR and Assembly PCR” In: The Encyclopedia of Molecular Biology.VCH Publishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’0 gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application serial no. 09/407,800, “SHUFFLING OF CODONALTERED GENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLECELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayreet al., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate proteases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high or lowtemperatures, and the like). Polypeptides encoded by the modifiednucleic acids can be screened for an activity before testing forproteolytic or other activity. Any testing modality or protocol can beused, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.6,361,974; 6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., aprotease or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)_(n) sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,proteases) molecules such that all 20 natural amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide (other aspectsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g. cloned into asuitable host, e.g., E. coli host, using, e.g., an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedproteolytic activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined -6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., proteases or antibodies of theinvention, with new or altered properties. SLR is a method of ligatingoligonucleotide fragments together non-stochastically. This methoddiffers from stochastic oligonucleotide shuffling in that the nucleicacid building blocks are not shuffled, concatenated or chimerizedrandomly, but rather are assembled non-stochastically. See, e.g., U.S.patent application Ser. No. 09/332,835 entitled “Synthetic LigationReassembly in Directed Evolution” and filed on Jun. 14, 1999 (“U.S. Ser.No. 09/332,835”). In one aspect, SLR comprises the following steps: (a)providing a template polynucleotide, wherein the template polynucleotidecomprises sequence encoding a homologous gene; (b) providing a pluralityof building block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10100 different chimeras. SLR can be used to generatelibraries comprised of over 101000 different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,proteases or antibodies of the invention, with new or alteredproperties. Optimized directed evolution is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionthat allow for the directed molecular evolution of nucleic acids throughrecombination. Optimized directed evolution allows generation of a largepopulation of evolved chimeric sequences, wherein the generatedpopulation is significantly enriched for sequences that have apredetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a 1/3 chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new protease phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., epoxide hydrolysis activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new protease phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,proteases, and the like.

In vivo shuffling can be performed utilizing the natural property ofcells to recombine multimers. While recombination in vivo has providedthe major natural route to molecular diversity, genetic recombinationremains a relatively complex process that involves 1) the recognition ofhomologies; 2) strand cleavage, strand invasion, and metabolic stepsleading to the production of recombinant chiasma; and finally 3) theresolution of chiasma into discrete recombined molecules. The formationof the chiasma requires the recognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide (e.g., a protease ofthe invention) and a second polynucleotide (e.g., an enzyme, such as aprotease of the invention or any other protease, or, a tag or anepitope). The invention can be used to produce a hybrid polynucleotideby introducing at least a first polynucleotide and a secondpolynucleotide which share at least one region of partial sequencehomology into a suitable host cell.

The regions of partial sequence homology promote processes which resultin sequence reorganization producing a hybrid polynucleotide. The term“hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., protease) sequences of theinvention. The invention also provides additional methods for isolatingproteases using the nucleic acids and polypeptides of the invention. Inone aspect, the invention provides for variants of a protease codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM DATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids areevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.

Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250 (see also discussion, above).

The invention also provides variants of polypeptides of the invention(e.g., proteases) comprising sequences in which one or more of the aminoacid residues (e.g., of an exemplary polypeptide of the invention) aresubstituted with a conserved or non-conserved amino acid residue (e.g.,a conserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code. Conservativesubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Thus,polypeptides of the invention include those with conservativesubstitutions of sequences of the invention, e.g., the exemplarypolypeptides of the invention, including but not limited to thefollowing replacements: replacements of an aliphatic amino acid such asAlanine, Valine, Leucine and Isoleucine with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue such as Aspartic acid and Glutamic acidwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asparagine and Glutamine, with another residue bearing anamide group; exchange of a basic residue such as Lysine and Argininewith another basic residue; and replacement of an aromatic residue suchas Phenylalanine, Tyrosine with another aromatic residue. Other variantsare those in which one or more of the amino acid residues of thepolypeptides of the invention includes a substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., protease activity, asdescribed herein. In other aspects, the variant, fragment, derivative,or analog includes a proprotein, such that the variant, fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying protease-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a protease toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding a protease modified to increase itsexpression in a host cell, protease so modified, and methods of makingthe modified proteases. The method comprises identifying a“non-preferred” or a “less preferred” codon in protease-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asStreptomyces diversa, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris, Bacillus subtilis. Exemplary host cells alsoinclude eukaryotic organisms, e.g., various yeast, such as Saccharomycessp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,Aspergillus niger, and mammalian cells and cell lines and insect cellsand cell lines. Thus, the invention also includes nucleic acids andpolypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding a protease isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the protease was derived, a yeast, a fungi, a plant cell, aninsect cell or a mammalian cell. Methods for optimizing codons are wellknown in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., a protease), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyprotease activity, or, as models to screen for agents that change theprotease activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing a protease of the invention, or, a fusion proteincomprising a protease of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a protease), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides plant products, e.g., oils, seeds, leaves, extracts andthe like, comprising a nucleic acid and/or a polypeptide (e.g., aprotease) of the invention. The transgenic plant can be dicotyledonous(a dicot) or monocotyledonous (a monocot). The invention also providesmethods of making and using these transgenic plants and seeds. Thetransgenic plant or plant cell expressing a polypeptide of the presentinvention may be constructed in accordance with any method known in theart. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's protease production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of protease. The can changeprotease activity in a plant. Alternatively, a protease of the inventioncan be used in production of a transgenic plant to produce a compoundnot naturally produced by that plant. This can lower production costs orcreate a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., aprotease) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., a protease orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′, 2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary polypeptide (amino acid) sequence of theinvention, e.g., proteins having a sequence as set forth in SEQ ID NO:2;SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ IDNO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ IDNO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ IDNO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ IDNO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ IDNO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ IDNO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ IDNO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ IDNO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ IDNO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO:122;SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ IDNO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159;SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ IDNO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 orSEQ ID NO:255, or the polypeptide encoded by SEQ ID NO:145. In oneaspect, the polypeptide has a protease activity, including proteinaseand/or peptidase activity, e.g., the ability to hydrolyze a peptidebond. The protease activity can comprise a peptidase activity, e.g., acarboxypeptidase activity, a dipeptidylpeptidase or an oligopeptidaseactivity, or an aminopeptidase activity. The protease activity cancomprise a serine proteinase activity, a metalloproteinase activity, acysteine protease activity and/or an aspartic protease activity, or, thesame or similar activity to a chymotrypsin, a trypsin, an elastase, akallikrein and/or a subtilisin.

Exemplary protease activities are set forth in Table 1, Table 2 andTable 3. Assays are described in detail in Examples, below. Assays weredeveloped to determine protease activity on a variety of pNA(para-nitroanalide) linked small peptide substrates as well as proteinsubstrates, such as casein, gelatin, corn zein, soybean trypsininhibitor, soybean lectin, and wheat germ lectin. For the small peptidesubstrate assays, hydrolysis of the terminal peptide bond liberates thepNA group and causes an increase in absorbance at 410 nm. To monitoractivity on the protein substrates, incubation of the protease andsubstrate at 37° C. was followed by monitoring the increase influorescence from an intramolecularly quenched substrate, byO-pthaldialdehyde (OPA) analysis, where in the presence of BME, OPAreacts with free amino ends to produce a fluorescent imidazole that canbe detected using a standard fluorescence plate reader, or by SDS-PAGEanalysis, where protease activity is indicated by the reduction ordisappearance of substrate band(s).

Proteinase activity on casein, gelatin, or corn zein was also determinedusing zymograms: zymogram gels contain the enzyme substrate (e.g.,alpha-zein) embedded within the gel matrix. If a protease has activityon the zein in the gel, a clearing zone will be produced within anotherwise blue background following electrophoresis, renaturation,development, and staining steps. The clearing zone corresponds to theposition of the protease in the gel.

Table 1, below, describes exemplary polypeptides having proteinaseactivity. SEQ ID NOS: Casein Gelatin AAA AAPF BAPNA GGF IEGR PFR 1 1,2 + + + + + + − − 2 7, 8 + + + + + + + − 3 3, 4 + − − − + − − 4 5,6 + + + + − − + − 5 29, 30 + + + + − + + + 6 49, 50 + + + + − + + + 723, 24 + + + + − − + + 8 65, 66 + + − + − − + + 9 43, 44 + + + + − − + +10 67, 68 + + − + − − − − 11 55, 56 + + + + − + + + 12 69, 70 + + + +− + + + 13 61, 62 + + − + − − + + 14 145, 146 + + + − − − + − 15 75,76 + + + + − − + + 16 31, 32 + + 17 143, 144 − + 18 27, 28 + + + + −− + + 19 79, 80 + + + + − + + Subtilisin A + + + + − + + −+ Indicates activity was detected on this substrate,− indicates that activity was not detected on this substrate using theconditions tested, and a blank box indicates that activity on thecorresponding substrate has yet to be determined.(AAPF = N-Suc-Alanine-Proline-Phenylalanine-pNA, AAA =N-Suc-Alanine-Alanine-Alanine-pNA, BAPNA = N-BZ = D,L-Arginine-pNA, GGF= N-Suc-Glycine-Glycine-Phenylalanine-pNA, IEGR =N-Suc-Isoleucine-Glutamate-Glycine-Arginine-pNA, PFR =N-Suc-Proline-Phenylalanine-Arginine-pNA).

Tables 2 and 3, below, describes exemplary polypeptides having peptidaseactivity, and summarizes their protease activities.

Table 2: TABLE 3 Activity summary OPA SDS-PAGE SEQ ID Zein SBTI SB WGZein SBTI SB WG AquaZe NOS: Lectin Lectin Lectin Lectin in Zymogram 7, 81.38 1.2 1.98 1.16 Yes Yes 69, 70 1.91 0.8 0.61 0.79 Yes M M Yes ND 65,66 3.59 .057* 0.48 0.88 Yes ND ND ND ND SEQ ID NOS: Casein Gelatin AAAAAPF BAPNA GGF IEGR PFR 9, 10 − − − − + − + + 15, 16 − − + − − − + 17,18 − − + + − − + + 85, 86 − − − − + + + + 63, 64 − − − + + + + + 57, 58− − + − − − + − 73, 74 1.73 0.5 0.23 0.61 Yes ND and 87, 88 29, 30 2.031.19 0.23 0.52 Yes M ND Yes Yes 23, 24 1.61 1.39 0.37 0.93 Yes M ND MYes 49, 50 1.38 0.88 0.45 0.92 Yes Yes 93, 94 1.49 1.11 0.24 0.95 M YesND ND M and 101, 102 103, 104 3.05 0.98 2.64 0.86 Yes Yes 41, 42 1.640.64 0.67 0.91 Yes Yes 19, 20 2.34 0.71 0.76 0.86 M Yes 77, 78 1.58 0.621.09 0.9 M M 31, 32 2.15 0.68 0.58 0.81 M ND ND ND Yes 67, 68 1.65 1.460.77 0.99 M ND ND ND Yes 61, 62 1.71 0.8 0.16 0.77 Yes ND ND ND ND 21,22 1.44 0.96 0.47 0.93 M ND 141, 142 1.78 1.04 0.71 1.07 ND Yes 1, 2 Yes43, 44 1.59* 0.86* 0.43* 0.8* ND ND ND ND YesND = no detectable activity under the conditions tested, M = Maybe(slight activity under the conditions tested)*Data from 48 hr time pointCorresponding negative controls were analyzed and shown to have nodetectable activity

OPA data is expressed as the ratio of the fluorescence (FL) of theenzyme and substrate reaction divided by the sum of the correspondingenzyme only and substrate only controls.${{Activity}\quad{Ratio}} = \frac{{FL}\quad{substrate}\quad{and}\quad{enzyme}\quad{preparation}\quad{reaction}}{\left( {{FL}\quad{substrate}\quad{alone}} \right) + \left( {{FL}\quad{enzyme}\quad{preparation}\quad{alone}} \right)}$

A fluorescence ratio of 1 indicates no activity above background. Afluorescence ratio above 1 indicates the presence of free amino endscreated by proteolysis of the substrate by the protease. An FL ratiobelow 1 may indicate that the protease is inhibited by the substratesuch that the hydrolysis of background proteins in the enzymepreparation occurs to a greater extent in the absence of the substratethan it does in the presence of substrate. In this case, the FLbackground fluorescence in the enzyme only control would be inflatedrelative to the background component of the enzyme and substrate sample.

The polypeptides of the invention include proteases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude proteases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains or active sites, of theprotease. In one aspect, the invention provides catalytic domains oractive sites as set forth below. In one aspect, the invention provides apeptide or polypeptide comprising or consisting of an active site domainas set forth below (the domains were predicted through use of thedatabase, Pfam, which is a large collection of multiple sequencealignments and hidden Markov models covering many common proteinfamilies, The Pfam protein families database, A. Bateman, E. Birney, L.Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L.Howe, M. Marshall, and E. L. L. Sonnhammer, Nucleic Acids Research,30(1):276-280, 2002):

SEQ ID NO: Domains (AA=Amino Acid)

248, 249AA(104) . . . (500)

-   -   Eukaryotic aspartyl protease    -   AA(112) . . . (317)

218, 219 Zinc carboxypeptdase

-   -   AA(116) . . . (325)

179, 180 Zinc carboxypeptidase

-   -   AA(117) . . . (321)

241, 242 Zinc carboxypeptidase

-   -   AA(121) . . . (228)    -   PA (protease associated) domain;    -   AA(234) . . . (468)

193, 194 Peptidase family M28

-   -   AA(124) . . . (340)

204, 205 Zinc carboxypeptidase

-   -   AA(124) . . . (344)

199, 200 Zinc carboxypeptidase

-   -   AA(128) . . . (378)

164, 165 Peptidase family M28

-   -   AA(156) . . . (426)    -   Subtilase family;    -   AA(74) . . . (142)

187, 188 Subtilisin N-terminal Region

-   -   AA(234) . . . (471)    -   Peptidase family M28;    -   AA(115) . . . (224)

222, 223 PA (protease associated) domain

-   -   AA(239) . . . (439)

171, 172 Peptidase family M48

-   -   AA(35) . . . (120)    -   Subtilisin N-terminal Region; AA(134) . . . (397)

229, 230 Subtilase family

-   -   AA(5) . . . (389)

150, 151 Eukaryotic aspartyl protease

-   -   AA(52) . . . (494)

210, 211 Serine carboxypeptidase

-   -   AA(74) . . . (522)

254, 255 Serine carboxypeptidase

-   -   AA(96) . . . (532)

158, 159 Serine carboxypeptidase

For example, the invention provides a peptide or polypeptide comprisingor consisting of an active site domain as set forth in residues 104 to500 of SEQ ID NO:249 (as encoded by SEQ ID NO:248), wherein the activesite has an aspartyl protease activity. In another aspect, the inventionprovides a peptide or polypeptide comprising or consisting of an activesite domain as set forth in residues 112 to 317 of SEQ ID NO:219 (asencoded by SEQ ID NO:218), wherein the active site has a zinccarboxypeptidase activity, etc.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences (e.g., see Table 4), prepro sequences andcatalytic domains (e.g., “active sites”) comprising sequences of theinvention. The percent sequence identity can be over the full length ofthe polypeptide, or, the identity can be over a region of at least about50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700 or more residues.

Polypeptides of the invention can also be shorter than the full lengthof exemplary polypeptides. In alternative aspects, the inventionprovides polypeptides (peptides, fragments) ranging in size betweenabout 5 and the full length of a polypeptide, e.g., an enzyme, such as aprotease; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g.,contiguous residues of an exemplary protease of the invention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, protease active sites (e.g., “catalyticdomains”), signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has aprotease activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention includes proteases of the invention with and withoutsignal. The polypeptide comprising a signal sequence of the invention(e.g., see Table 4) can be a protease of the invention or anotherprotease or another enzyme or other polypeptide.

The invention includes immobilized proteases, anti-protease antibodiesand fragments thereof. The invention provides methods for inhibitingprotease activity, e.g., using dominant negative mutants oranti-protease antibodies of the invention. The invention includesheterocomplexes, e.g., fusion proteins, heterodimers, etc., comprisingthe proteases of the invention.

Polypeptides of the invention can have a protease activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativeprotease preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, protease variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofprotease variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify protease modulators, e.g., activators or inhibitors of proteaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to protease assays to determine their ability to inhibitsubstrate cleavage. Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith proteases, inhibitors can be combined to increase the spectrum ofactivity.

The enzymes of the invention are also useful as research reagents todigest proteins or in protein sequencing. For example, the proteases maybe used to break polypeptides into smaller fragments for sequencingusing, e.g. an automated sequencer.

The invention also provides methods of discovering new proteases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, phagemid libraries are screened for expression-based discoveryof proteases In another aspect, lambda phage libraries are screened forexpression-based discovery of proteases. Screening of the phage orphagemid libraries can allow the detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of phage or phagemidlibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

The present invention includes protease enzymes which are non-naturallyoccurring carbonyl hydrolase variants (e.g., protease variants) having adifferent proteolytic activity, stability, substrate specificity, pHprofile and/or performance characteristic as compared to the precursorcarbonyl hydrolase from which the amino acid sequence of the variant isderived. Specifically, such protease variants have an amino acidsequence not found in nature, which is derived by substitution of aplurality of amino acid residues of a precursor protease with differentamino acids. The precursor protease may be a naturally-occurringprotease or a recombinant protease. The useful protease variantsencompass the substitution of any of the naturally occurring L-aminoacids at the designated amino acid residue positions.

Protease Signal Sequences, Prepro and Catalytic Domains

The invention provides protease signal sequences (e.g., signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs of the invention can be isolated or recombinantpeptides or can be part of a fusion protein, e.g., as a heterologousdomain in a chimeric protein. The invention provides nucleic acidsencoding these catalytic domains (CDs), prepro domains and signalsequences (SPs, e.g., a peptide having a sequence comprising/consistingof amino terminal residues of a polypeptide of the invention).

In one aspect, the invention provides a signal sequence comprising apeptide comprising/consisting of a sequence as set forth in residues 1to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1to 44 (or a longer peptide) of a polypeptide of the invention.

In an alternative aspect, the invention provides a signal sequencecomprising a peptide comprising/consisting of a sequence as set forth inTable 4, below: SEQ ID NO: Signal (AA) 1, 2 1-37 101, 102 1-22 111, 1121-36 113, 114 1-32 115, 116 1-33 121, 122 1-25 123, 124 1-56 127, 1281-27 13, 14 1-33 131, 132 1-21 133, 134 1-27 139, 140 1-38 141, 142 1-25143, 144 1-35 15, 16 1-31 164, 165 1-17 179, 180 1-21 19, 20 1-39 193,194 1-19 199, 200 1-18 21, 22 1-22 210, 211 1-19 222, 223 1-15 229, 2301-21 23, 24 1-23 241, 242 1-20 254, 255 1-18 27, 28 1-27 29, 30 1-24 3,4 1-36 31, 32 1-26 35, 36 1-27 37, 38 1-37 41, 42 1-22 43, 44 1-25 45,46 1-26 47, 48 1-24 49, 50 1-30 5, 6 1-32 51, 52 1-27 53, 54 1-32 55, 561-27 57, 58 1-31 61, 62 1-40 67, 68 1-27 69, 70 1-32 71, 72 1-25 73, 741-28 75, 76 1-25 81, 82 1-20 83, 84 1-22 85, 86 1-20 87, 88 1-35 89, 901-32  9, 10 1-28 93, 94 1-36 95, 96 1-24

The protease signal sequences (SPs) and/or prepro sequences of theinvention can be isolated peptides, or, sequences joined to anotherprotease or a non-protease polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisingprotease signal sequences of the invention. In one aspect, polypeptidescomprising protease signal sequences SPs and/or prepro of the inventioncomprise sequences heterologous to a protease of the invention (e.g., afusion protein comprising an SP and/or prepro of the invention andsequences from another protease or a non-protease protein). In oneaspect, the invention provides proteases of the invention withheterologous SPs and/or prepro sequences, e.g., sequences with a yeastsignal sequence. A protease of the invention can comprise a heterologousSP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen,Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel protease polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom 13 to 36 amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel protease signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects proteases of the inventionmay not have SPs and/or prepro sequences, or “domains.” In one aspect,the invention provides the proteases of the invention lacking all orpart of an SP and/or a prepro domain. In one aspect, the inventionprovides a nucleic acid sequence encoding a signal sequence (SP) and/orprepro from one protease operably linked to a nucleic acid sequence of adifferent protease or, optionally, a signal sequence (SPs) and/or preprodomain from a non-protease protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa protease) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated or recombinant polypeptide comprising (orconsisting of) a polypeptide comprising a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention with the provisothat it is not associated with any sequence to which it is naturallyassociated (e.g., a protease sequence). Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

Hybrid (Chimeric) Proteases and Peptide Libraries

In one aspect, the invention provides hybrid proteases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asprotease substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like. In one aspect,the invention provides chimeric proteins comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention or acombination thereof and a heterologous sequence (see above).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of proteases of the invention and other peptides, includingknown and random peptides. They can be fused in such a manner that thestructure of the proteases is not significantly perturbed and thepeptide is metabolically or structurally conformationally stabilized.This allows the creation of a peptide library that is easily monitoredboth for its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of a protease sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined.

For example, in order to optimize the performance of a mutation at agiven site, random mutagenesis may be conducted at the target codon orregion and the expressed protease variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, as discussed herein for example, M13 primer mutagenesis and PCRmutagenesis.

Screening of the mutants can be done using assays of proteolyticactivities. In alternative aspects, amino acid substitutions can besingle residues; insertions can be on the order of from about 1 to 20amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides proteases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. protease activity) although variants can be selected tomodify the characteristics of the proteases as needed.

In one aspect, proteases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the proteases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the protease are linkedtogether, in such a manner as to minimmize the disruption to thestability of the protease structure, e.g., it retains protease activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forprotease activity (e.g., assays such as hydrolysis of casein inzymograms, the release of fluorescence from gelatin, or the release ofp-nitroanalide from various small peptide substrates), to screencompounds as potential modulators, e.g., activators or inhibitors, of aprotease activity, for antibodies that bind to a polypeptide of theinvention, for nucleic acids that hybridize to a nucleic acid of theinvention, to screen for cells expressing a polypeptide of the inventionand the like. In addition to the array formats described in detail belowfor screening samples, alternative formats can also be used to practicethe methods of the invention. Such formats include, for example, massspectrometers, chromatographs, e.g., high-throughput HPLC and otherforms of liquid chromatography, and smaller formats, such as 1536-wellplates, 384-well plates and so on. High throughput screening apparatuscan be adapted and used to practice the methods of the invention, see,e.g., U.S. Patent Application No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a protease gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a protease of the invention. These antibodies canbe used to isolate, identify or quantify the proteases of the inventionor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related proteases.The antibodies can be designed to bind to an active site of a protease.Thus, the invention provides methods of inhibiting proteases using theantibodies of the invention (see discussion above regarding applicationsfor anti-protease compositions of the invention).

The invention provides fragments of the enzymes of the invention,including immunogenic fragments of a polypeptide of the invention, e.g.,SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ IDNO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ IDNO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ IDNO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ IDNO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ IDNO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ IDNO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ IDNO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ IDNO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ IDNO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ IDNO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120;SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ IDNO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151;SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ IDNO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242;SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ IDNO:145. The immunogenic peptides of the invention (e.g., the immunogenicfragments of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ IDNO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ IDNO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ IDNO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ IDNO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ IDNO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ IDNO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ IDNO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ IDNO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ IDNO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ IDNO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118;SEQ ID NO:120; SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ IDNO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147;SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ IDNO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235;SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255, or the polypeptideencoded by SEQ ID NO:145) can further comprise adjuvants, carriers andthe like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the proteases, of the invention.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides (e.g., proteases) from otherorganisms and samples. In such techniques, polypeptides from theorganism are contacted with the antibody and those polypeptides whichspecifically bind the antibody are detected. Any of the proceduresdescribed above may be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., proteases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified protease activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention, e.g., acoding sequence for an enzyme of the invention. See, e.g., WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the proteases of the invention.

Metabolic flux analysis (MA) is based on a known biochemistry framework.A linearly independent metabolic matrix is constructed based on the lawof mass conservation and on the pseudo-steady state hypothesis (PSSH) onthe intracellular metabolites. In practicing the methods of theinvention, metabolic networks are established, including the:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., aprotease message) or generating new (e.g., protease) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of a protease of the invention or by protease activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., aprotease) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of proteasepresent or by protease activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

INDUSTRIAL APPLICATIONS

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides (e.g., proteases) of the invention, and methods of makingand using these compositions. The invention incorporates all methods ofmaking and using detergent compositions, see, e.g., U.S. Pat. Nos.6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent compositionscan be a one and two part aqueous composition, a non-aqueous liquidcomposition, a cast solid, a granular form, a particulate form, acompressed tablet, a gel and/or a paste and a slurry form. The proteasesof the invention can also be used as a detergent additive product in asolid or a liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

The invention also provides methods capable of removing gross foodsoils, films of food residue and other minor food compositions usingthese detergent compositions. Proteases of the invention can facilitatethe removal of stains by means of catalytic hydrolysis of proteins.Proteases of the invention can be used in dishwashing detergents intextile laundering detergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof protease present in the final solution ranges from about 0.001 mg to0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the proteases of thepresent invention are active in the pH ranges of from about 4 to about12 and in the temperature range of from about 20° C. to about 95° C. Thedetergents of the invention can comprise cationic, semi-polar nonionicor zwitterionic surfactants; or, mixtures thereof.

Proteases of the invention can be formulated into powdered and liquiddetergents having pH between 4.0 and 12.0 at levels of about 0.01 toabout 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as proteases,cellulases, lipases or endoglycosidases, endo-beta.-1,4-glucanases,beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent compositions can also include builders and stabilizers.

The addition of proteases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe compositions of the invention as long as the enzyme is active at ortolerant of the pH and/or temperature of the intended use. In addition,the proteases of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A protease of the invention maybe included as a detergent additive. The detergent composition of theinvention may, for example, be formulated as a hand or machine laundrydetergent composition comprising a polypeptide of the invention. Alaundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a protease of the invention. Alternatively, a protease ofthe invention can be formulated as a detergent composition for use ingeneral household hard surface cleaning operations. In alternativeaspects, detergent additives and detergent compositions of the inventionmay comprise one or more other enzymes such as a protease, a lipase, acutinase, another protease, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alactase, and/or a peroxidase (see also, above). The properties of theenzyme(s) of the invention are chosen to be compatible with the selecteddetergent (i.e. pH-optimum, compatibility with other enzymatic andnon-enzymatic ingredients, etc.) and the enzyme(s) is present ineffective amounts. In one aspect, protease enzymes of the invention areused to remove malodorous materials from fabrics. Various detergentcompositions and methods for making them that can be used in practicingthe invention are described in, e.g., U.S. Pat. Nos. 6,333,301;6,329,333; 6,326,341; 6,297,038; 6,309,871; 6,204,232; 6,197,070;5,856,164.

When formulated as compositions suitable for use in a laundry machinewashing method, the proteases of the invention can comprise both asurfactant and a builder compound. They can additionally comprise one ormore detergent components, e.g., organic polymeric compounds, bleachingagents, additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. Laundry compositions of the invention can also containsoftening agents, as additional detergent components. Such compositionscontaining carbohydrase can provide fabric cleaning, stain removal,whiteness maintenance, softening, color appearance, dye transferinhibition and sanitization when formulated as laundry detergentcompositions.

The density of the laundry detergent compositions of the invention canrange from about 200 to 1500 g/liter, or, about 400 to 1200 g/liter, or,about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition; thiscan be measured at about 20° C.

The “compact” form of laundry detergent compositions of the invention isbest reflected by density and, in terms of composition, by the amount ofinorganic filler salt. Inorganic filler salts are conventionalingredients of detergent compositions in powder form. In conventionaldetergent compositions, the filler salts are present in substantialamounts, typically 17% to 35% by weight of the total composition. In oneaspect of the compact compositions, the filler salt is present inamounts not exceeding 15% of the total composition, or, not exceeding10%, or, not exceeding 5% by weight of the composition. The inorganicfiller salts can be selected from the alkali and alkaline-earth-metalsalts of sulphates and chlorides, e.g., sodium sulphate.

Liquid detergent compositions of the invention can also be in a“concentrated form.” In one aspect, the liquid detergent compositionscan contain a lower amount of water, compared to conventional liquiddetergents. In alternative aspects, the water content of theconcentrated liquid detergent is less than 40%, or, less than 30%, or,less than 20% by weight of the detergent composition. Detergentcompounds of the invention can comprise formulations as described in WO97/01629.

Proteases, such as metalloproteases (MPs) and serine proteases, of theinvention can be useful in formulating various cleaning compositions. Anumber of known compounds are suitable surfactants including nonionic,anionic, cationic, or zwitterionic detergents, can be used, e.g., asdisclosed in U.S. Pat. Nos. 4,404,128; 4,261,868; 5,204,015. Inaddition, proteases can be used, for example, in bar or liquid soapapplications, dish care formulations, contact lens cleaning solutions orproducts, peptide hydrolysis, waste treatment, textile applications, asfusion-cleavage enzymes in protein production, and the like. Proteasesmay provide enhanced performance in a detergent composition as comparedto another detergent protease, that is, the enzyme group may increasecleaning of certain enzyme sensitive stains such as grass or blood, asdetermined by usual evaluation after a standard wash cycle.Metalloproteases, serine proteases (or other proteases of the invention)can be formulated into known powdered and liquid detergents having pHbetween 6.5 and 12.0 at levels of about 0.01 to about 5% (for example,about 0.1% to 0.5%) by weight. These detergent cleaning compositions canalso include other enzymes such as known proteases, amylases,cellulases, lipases or endoglycosidases, as well as builders andstabilizers.

Treating Fibers and Textiles

The invention provides methods of treating fibers and fabrics using oneor more proteases of the invention. The proteases can be used in anyfiber- or fabric-treating method, which are well known in the art, see,e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536;6,017,751; 5,980,581; US Patent Publication No. 20020142438 A1. Forexample, proteases of the invention can be used in fiber and/or fabricdesizing. In one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a protease of theinvention in a solution. In one aspect, the fabric is treated with thesolution under pressure. For example, proteases of the invention can beused in the removal of stains.

In one aspect, proteases of the invention are applied during or afterthe weaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The proteases of the invention can be applied toremove these sizing starch or starch derivatives. After the textileshave been woven, a fabric can proceed to a desizing stage. This can befollowed by one or more additional fabric processing steps. Desizing isthe act of removing “size” from textiles. After weaving, the sizecoating must be removed before further processing the fabric in order toensure a homogeneous and wash-proof result. The invention provides amethod of desizing comprising enzymatic treatment of the “size” by theaction of proteases of the invention.

The enzymes of the invention can be used to desize fabrics, includingcotton-containing fabrics, as detergent additives, e.g., in aqueouscompositions. The invention provides methods for producing a stonewashedlook on indigo-dyed denim fabric and garments. For the manufacture ofclothes, the fabric can be cut and sewn into clothes or garments. Thesecan be finished before or after the treatment. In particular, for themanufacture of denim jeans, different enzymatic finishing methods havebeen developed. The finishing of denim garment normally is initiatedwith an enzymatic desizing step, during which garments are subjected tothe action of amylolytic enzymes in order to provide softness to thefabric and make the cotton more accessible to the subsequent enzymaticfinishing steps. The invention provides methods of finishing denimgarments (e.g., a “bio-stoning process”), enzymatic desizing andproviding softness to fabrics using the proteases of the invention. Theinvention provides methods for quickly softening denim garments in adesizing and/or finishing process.

Other enzymes can be also be used in these desizing processes. Forexample, an alkaline and thermostable amylase and protease can becombined in a single bath for desizing and bioscouring. Among advantagesof combining desizing and scouring in one step are cost reduction andlower environmental impact due to savings in energy and water usage andlower waste production. Exemplary application conditions for desizingand bioscouring are about pH 8.5 to 10.0 and temperatures of about 40°C. and up. Using a protease of the invention, low enzyme dosages, e.g.,about 100 grams (g) per a ton of cotton, and short reaction times, e.g.,about 15 minutes, can be used to obtain efficient desizing and scouringwith out added calcium.

In one aspect, an alkaline and thermostable amylase and protease arecombined in a single bath desizing and bioscouring. Among advantages ofcombining desizing and scouring in one step are cost reduction and lowerenvironmental impact due to savings in energy and water usage and lowerwaste production. Application conditions for desizing and bioscouringcan be between about pH 8.5 to pH 10.0 and temperatures at about 40° C.and up. Low enzyme dosages (e.g., about 100 g per a ton of cotton) andshort reaction times (e.g., about 15 minutes) can be used to obtainefficient desizing and scouring with out added calcium.

The proteases of the invention can be used in combination with othercarbohydrate degrading enzymes, e.g., cellulase, arabinanase,xyloglucanase, pectinase, and the like, for the preparation of fibers orfor cleaning of fibers. These can be used in combination withdetergents. In one aspect, proteases of the invention can be used intreatments to prevent the graying of a textile.

The proteases of the invention can be used to treat any cellulosicmaterial, including fibers (e.g., fibers from cotton, hemp, flax orlinen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g. originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or is rayon/viscose with one or more companion material suchas wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The textile treating processes of the invention (using proteases of theinvention) can be used in conjunction with other textile treatments,e.g., scouring and bleaching. Scouring is the removal of non-cellulosicmaterial from the cotton fiber, e.g., the cuticle (mainly consisting ofwaxes) and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability. This is needed for dyeing. Removal of the primary cellwalls by the processes of the invention improves wax removal and ensuresa more even dyeing. Treating textiles with the processes of theinvention can improve whiteness in the bleaching process. The mainchemical used in scouring is sodium, hydroxide in high concentrationsand at high temperatures. Bleaching comprises oxidizing the textile.Bleaching typically involves use of hydrogen peroxide as the oxidizingagent in order to obtain either a fully bleached (white) fabric or toensure a clean shade of the dye.

The invention also provides alkaline proteases (proteases active underalkaline conditions). These have wide-ranging applications in textileprocessing, degumming of plant fibers (e.g., plant bast fibers),treatment of pectic wastewaters, paper-making, and coffee and teafermentations. See, e.g., Hoondal (2002) Applied Microbiology andBiotechnology 59:409-418.

Treating Foods and Food Processing

The proteases of the invention have numerous applications in foodprocessing industry. For example, in one aspect, the proteases of theinvention are used to improve the extraction of oil from oil-rich plantmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans,olive oil from olives, rapeseed oil from rapeseed and/or sunflower oilfrom sunflower seeds.

The proteases of the invention can be used for separation of componentsof plant cell materials. For example, proteases of the invention can beused in the separation of protein-rich material (e.g., plant cells) intocomponents, e.g., sucrose from sugar beet or starch or sugars frompotato, pulp or hull fractions. In one aspect, proteases of theinvention can be used to separate protein-rich or oil-rich crops intovaluable protein and oil and hull fractions. The separation process maybe performed by use of methods known in the art.

The proteases of the invention can be used in the preparation of fruitor vegetable juices, syrups, extracts and the like to increase yield.The proteases of the invention can be used in the enzymatic treatment(e.g., hydrolysis of proteins) of various plant cell wall-derivedmaterials or waste materials, e.g. from wine or juice production, oragricultural residues such as vegetable hulls, bean hulls, sugar beetpulp, olive pulp, potato pulp, and the like. The proteases of theinvention can be used to modify the consistency and appearance ofprocessed fruit or vegetables. The proteases of the invention can beused to treat plant material to facilitate processing of plant material,including foods, facilitate purification or extraction of planetcomponents. The proteases of the invention can be used to improve feedvalue, decrease the water binding capacity, improve the degradability inwaste water plants and/or improve the conversion of plant material toensilage, and the like.

Animal Feeds and Food or Feed Additives

The invention provides methods for treating animal feeds and foods andfood or feed additives using proteases of the invention, animalsincluding mammals (e.g., humans), birds, fish and the like. Theinvention provides animal feeds, foods, and additives comprisingproteases of the invention. In one aspect, treating animal feeds, foodsand additives using proteases of the invention can help in theavailability of nutrients, e.g., starch, in the animal feed or additive.By breaking down difficult to digest proteins or indirectly or directlyunmasking starch (or other nutrients), the protease makes nutrients moreaccessible to other endogenous or exogenous enzymes. The protease canalso simply cause the release of readily digestible and easily absorbednutrients and sugars.

Proteases of the present invention, in the modification of animal feedor a food, can process the food or feed either in vitro (by modifyingcomponents of the feed or food) or in vivo. Proteases can be added toanimal feed or food compositions containing high amounts ofarabinogalactans or galactans, e.g. feed or food containing plantmaterial from soy bean, rape seed, lupin and the like. When added to thefeed or food the protease significantly improves the in vivo break-downof plant cell wall material, whereby a better utilization of the plantnutrients by the animal (e.g., human) is achieved. In one aspect, thegrowth rate and/or feed conversion ratio (i.e. the weight of ingestedfeed relative to weight gain) of the animal is improved. For example apartially or indigestible galactan-comprising protein is fully orpartially degraded by a protease of the invention, e.g. in combinationwith another enzyme, e.g., beta-galactosidase, to peptides and galactoseand/or galactooligomers. These enzyme digestion products are moredigestible by the animal. Thus, proteases of the invention cancontribute to the available energy of the feed or food. Also, bycontributing to the degradation of galactan-comprising proteins, aprotease of the invention can improve the digestibility and uptake ofcarbohydrate and non-carbohydrate feed or food constituents such asprotein, fat and minerals.

In another aspect, protease of the invention can be supplied byexpressing the enzymes directly in transgenic feed crops (as, e.g.,transgenic plants, seeds and the like), such as corn, soy bean, rapeseed, lupin and the like. As discussed above, the invention providestransgenic plants, plant parts and plant cells comprising a nucleic acidsequence encoding a polypeptide of the invention. In one aspect, thenucleic acid is expressed such that the protease of the invention isproduced in recoverable quantities. The protease can be recovered fromany plant or plant part. Alternatively, the plant or plant partcontaining the recombinant polypeptide can be used as such for improvingthe quality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor.

Paper or Pulp Treatment

The proteases of the invention can be in paper or pulp treatment orpaper deinking. For example, in one aspect, the invention provides apaper treatment process using proteases of the invention. In anotheraspect, paper components of recycled photocopied paper during chemicaland enzymatic deinking processes. In one aspect, proteases of theinvention can be used in combination with cellulases, pectate lyases orother enzymes. The paper can be treated by the following threeprocesses: 1) disintegration in the presence of proteases of theinvention, 2) disintegration with a deinking chemical and proteases ofthe invention, and/or 3) disintegration after soaking with proteases ofthe invention. The recycled paper treated with proteases can have ahigher brightness due to removal of toner particles as compared to thepaper treated with just cellulase. While the invention is not limited byany particular mechanism, the effect of proteases of the invention maybe due to its behavior as surface-active agents in pulp suspension.

The invention provides methods of treating paper and paper pulp usingone or more proteases of the invention. The proteases of the inventioncan be used in any paper- or pulp-treating method, which are well knownin the art, see, e.g., U.S. Pat. Nos. 6,241,849; 6,066,233; 5,582,681.For example, in one aspect, the invention provides a method for deinkingand decolorizing a printed paper containing a dye, comprising pulping aprinted paper to obtain a pulp slurry, and dislodging an ink from thepulp slurry in the presence of proteases of the invention (other enzymescan also be added). In another aspect, the invention provides a methodfor enhancing the freeness of pulp, e.g., pulp made from secondaryfiber, by adding an enzymatic mixture comprising proteases of theinvention (can also include other enzymes, e.g., pectate lyase,cellulase, amylase or glucoamylase enzymes) to the pulp and treatingunder conditions to cause a reaction to produce an enzymatically treatedpulp. The freeness of the enzymatically treated pulp is increased fromthe initial freeness of the secondary fiber pulp without a loss inbrightness.

Waste Treatment

The proteases of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingproteases of the invention. The methods can comprise reducing the massand volume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including proteases of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

In addition, the proteases of the invention can be used in the animalrendering industry, to e.g., get rid of feathers, e.g., as described byYamamura (2002) Biochem. Biophys. Res. Com. 294:1138-1143. Alkalineproteases can also be used in the production of proteinaceous fodderfrom waste feathers or keratin-containing materials, e.g., as describedby Gupta (2002) Appl. Microbiol. Biotechnol. 59:15-32.

Oral Care Products

The invention provides oral care product comprising proteases of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising proteases of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. A protease of the invention is used at any point in thefermentation process. For example, proteases of the invention can beused in the processing of barley malt. The major raw material of beerbrewing is barley malt This can be a three stage process. First, thebarley grain can be steeped to increase water content, e.g., to aroundabout 40%. Second, the grain can be germinated by incubation at 15 to25° C. for 3 to 6 days when enzyme synthesis is stimulated under thecontrol of gibberellins. In one aspect, proteases of the invention areadded at this (or any other) stage of the process. The action ofproteases results in an increase in fermentable reducing sugars. Thiscan be expressed as the diastatic power, DP, which can rise from around80 to 190 in 5 days at 12° C. Proteases of the invention can be used inany beer or alcoholic beverage producing process, as described, e.g., inU.S. Pat. No. 5,762,991; 5,536,650; 5,405,624; 5,021,246; 4,788,066.

Medical and Research Applications

Proteases of the invention can be used for cell isolation from tissuefor cellular therapies in the same manner that collagenases. Forexample, metallo-endoproteinases and other enzymes of the invention thatcan cleave collagen into smaller peptide fragments, can be used as“liberase enzymes” for tissue dissociation and to improve the health ofisolated cells. “Liberase enzymes” can replace traditional collagenase.Proteases of the invention having collagenase I, collagenase II,clostripain and/or neutral protease activity can be used for tissuedissociation. In one aspect, for tissue dissociation, collagenaseisoforms of the invention are blended with each other, and, optionally,with a neutral protease. In one aspect, the neutral protease is aneutral protease dispase and/or the neutral protease thermolysin.

Additionally, proteases of the invention can be used as antimicrobialagents, due to their bacteriolytic properties, as described, e.g., inLi, S. et. al. Bacteriolytic Activity and Specificity of Achromobacterb-Lytic Protease, J. Biochem. 124, 332-339 (1998).

Proteases of the invention can also be used therapeutically to cleaveand destroy specific proteins. Potential targets include toxin proteins,such as Anthrax, Clostridium botulinum, Ricin, and essential viral orcancer cell proteins.

Proteases of the invention can also be used in disinfectants, asdescribed, e.g., in J. Gen Microbiol (1991) 137(5): 1145-1153; Science(2001) 249:2170-2172.

Additional medical uses of the proteases of the invention include lipomaremoval, wound debraidment and scar prevention (collagenases), debridingchronic dermal ulcers and severely burned areas.

Proteases of the invention can be used to in sterile enzymatic debridingcompositions, e.g., ointments, in one aspect, containing about 250collagenase units per gram. White petrolatum USP can be a carrier. Inone aspect, proteases of the invention can be used in indicationssimilar to Santyl® Ointment (BTC, Lynbrook, NY). Proteases of theinvention can also be used in alginate dressings, antimicrobial barrierdressings, burn dressings, compression bandages, diagnostic tools, geldressings, hydro-selective dressings, hydrocellular (foam) dressings,hydrocolloid Dressings, I.V dressings, incise drapes, low adherentdressings, odor absorbing dressings, paste bandages, post operativedressings, scar management, skin care, transparent film dressings and/orwound closure. Proteases of the invention can be used in woundcleansing, wound bed preparation, to treat pressure ulcers, leg ulcers,burns, diabetic foot ulcers, scars, IV fixation, surgical wounds andminor wounds.

Additionally, proteases of the invention can be used in proteomics andlab work in general. For instance, proteases can be used in the samemanner as DNA restriction enzymes.

Other Industrial Applications

The invention also includes a method of increasing the flow ofproduction fluids from a subterranean formation by removing a viscous,protein-containing, damaging fluid formed during production operationsand found within the subterranean formation which surrounds a completedwell bore comprising allowing production fluids to flow from the wellbore; reducing the flow of production fluids from the formation belowexpected flow rates; formulating an enzyme treatment by blendingtogether an aqueous fluid and a polypeptide of the invention; pumpingthe enzyme treatment to a desired location within the well bore;allowing the enzyme treatment to degrade the viscous,protein-containing, damaging fluid, whereby the fluid can be removedfrom the subterranean formation to the well surface; and wherein theenzyme treatment is effective to attack protein in cell walls.

Proteases of the invention can be used for peptide synthesis, in theleather industry, e.g., for hide processing, e.g., in hair removaland/or bating, for waste management, e.g., removal of hair from drains,in the photography industry, e.g., for silver recovery from film, in themedical industry, e.g., as discussed above, e.g., for treatment ofburns, wounds, carbuncles, furuncles and deep abscesses or to dissolveblood clots by dissolving fibrin, for silk degumming.

In other aspects, proteases of the invention can be used as flavorenhancers in, for example, cheese and pet food, as described, e.g., inPommer, K., Investigating the impact of enzymes on pet foodpalatability, Petfood Industry, May 2002, 10-11.

In yet another embodiment of the invention, proteases of the inventioncan be used to increase starch yield from corn wet milling, asdescribed, e.g., in Johnston, D. B., and Singh, V. Use of proteases toReduce Steep Time and SO2 requirements in a corn wet-milling process,Cereal Chem. 78(4):405-411.

In other aspects, proteases of the invention can be used in biodefense(e.g., destruction of spores or bacteria). Use of proteases inbiodefense applications offer a significant benefit, in that they can bevery rapidly developed against any currently unknown biological warfareagents of the future. In addition, proteases of the invention can beused for decontamination of affected environments.

Additionally, proteases of the invention can be used in biofilmdegradation, in biomass conversion to ethanol, and/or in the personalcare and cosmetics industry.

Proteases of the invention can also be used to enhanceenantioselectivity, as described, e.g., in Arisawa, A. et. al.Streptomyces Serine Protease (DHP-A) as a New Biocatalyst Capable ofForming Chiral Intermediates of 1,4-Diohydropyridine CalciumAntagonists. Appl Environ Mircrobiol 2002 June; 68(6):2716-2725; Haring,D. et. al. Semisynthetic Enzymes in AsymmetricSynthesis:Enantioselective Reduction of Racemic Hydroperoxides Catalyzedby Seleno-Subtilisin. J. Org. Chem. 1999, 64:832-835.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Protease Activity Assays

The following example describes exemplary protease activity assays todetermine the catalytic activity of a protease. These exemplary assayscan be used to determine if a polypeptide is within the scope of theinvention.

The activity assays used for proteinases (active on proteins) includezymograms and liquid substrate enzyme assays. Three different types ofzymograms were used to measure activity: casein, gelatin and zein. Forthe liquid substrate enzyme assays, three main types were used: gelelectrophoresis, O-pthaldialdehyde (OPA), and fluorescent end pointassays. For both the gel electrophoresis and OPA assays, four differentsubstrates were used: zein, Soybean Trypsin Inhibitor (SBTI,SIGMA-Aldrich, T6522), wheat germ lectin and soybean lectin. Thesubstrate for the fluorescent end point assay was gelatin.

The activity assays used for proteinases and peptidases (active onpeptides) used pNA linked small peptide substrates. The assays includedspecificity end point assays, unit definition kinetic assays and pHassays.

The following example describes the above-mentioned exemplary proteaseactivity assays. These exemplary assays can be used to determine if apolypeptide is within the scope of the invention.

Protein (Proteinase Activity)

Casein Zymogram Gel Assays

Casein zymogram gels were used to assess proteinase activity (see Tables1 and 2). The protease activity assays were assessed using 4-16%gradient gels (Invitrogen Corp., Carlsbad, Calif.) containing caseinconjugated to a blue dye and embedded within the gel matrix. Allzymogram gels were processed according to the manufacturer'sinstructions. Briefly, each sample was mixed with an equal volume of 2×loading dye and incubated without heating for ten minutes beforeloading. After electrophoresis, gels were incubated in a renaturingbuffer to remove the SDS and allow the proteins to regain their nativeform. Gels were then transferred to a developing solution and incubatedat 37° C. for 4 to 24 hours. If a protease digests the casein in thegel, a clear zone is produced against the otherwise blue background thatcorresponds to the location of the protease in the gel. Negativecontrols (indicated with NC on gel images) were processed along with theexperimental samples in each experiment and electrophoresed on thecasein zymograms next to their corresponding protease(s).

Unlike traditional SDS-PAGE, samples are not heat denatured prior toelectrophoresis of casein zymograms. As a result, it is sometimesdifficult to accurately assess the molecular weight of the proteases.For example, Subtilisin A (Sigma, P5380, indicated with Subt.A on thegel images), which was used as a positive control in these experiments,is predicted to be approximately 27 kDa in size. However, whenelectrophoresed through casein zymograms using the conditions described,Subtilisin A barely migrates into the gel and is visible only above 183kDa. Therefore, the zymograms do not define the MW of the proteasesindicated, but rather used as an indicator of activity.

Gelatin Zymogram Assays

Gelatin zymograms, Novex® Zymogram Gels, were performed according tomanufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.). Unlikethe casein zymograms, gelatin zymograms were post-stained followingdevelopment using either a Colloidal Blue Staining Kit or the SIMLYBLUE™Safestain, (both from Invitrogen). Areas of protease activity appearedas clear bands against a dark background.

Corn Zein Assays

Corn zein was used as substrate for protease activity assays, usingpowder, Z-3625 (Sigma Chemical Co. St. Louis, Mo.), and Aquazein, 10%solution (Freeman Industries, Tuckahoe, N.Y.). When fractionated througha SDS-PAGE gel, zein from both suppliers produced bands of 24 and 22kDa. The two zein bands correspond in molecular weight to thosepreviously described for alpha-zein, the most abundant subclass ofzeins, which are estimated to comprise 71-84% of total zein in corn(see, e.g., Consoli (2001) Electrophoresis 22:2983-2989). Results areillustrated in Table 3, above.

Lyophilized culture supernatants containing active protease wereresuspended, dialyzed, and incubated with zein in 50 mM KPO₄, pH 7.5.Reactions were run in a 96-well microtiter format. “Substrate only” and“enzyme preparation only” controls were processed as well asexperimental samples. After 24 hours at 30° C., aliquots were removedand subjected to OPA, SDS-PAGE, or Zymogram analysis. In some cases,fresh aliquots were removed and analyzed after 48 or 72 hours at 30° C.

Zein Zymogram: Aquazein was added to a final concentration of 0.075% ina 10% polyacrylamide gel. Aliquots of dialyzed protease samples wereelectrophoresed through the zein zymogram using standard conditions.Following electrophoresis, the zymogram gel was washed, incubated in arenaturing buffer, incubated overnight in a developing buffer optimizedfor protease activity (contains NaCl, CaCl₂, and Brij 35, in Tris bufferpH 8), and stained with Coomassie blue stain.

SDS-PAGE: Aliquots of equal volume were removed from each sample andsubjected to SDS-PAGE analysis. Following electrophoresis, proteins inthe gels were stained with SYPRO Orange (Molecular Probes) andvisualized using UV transillumination.

OPA: In the presence of Beta-mercaptoethanol (BME), OPA reacts with freeamino ends to produce a fluorescent imidazole that can be detected usinga standard fluorescence plate reader. In this assay, aliquots of equalvolume were removed from each sample and placed in a black fluorescenceplate. Samples were then diluted 1:10 in OPA reagents. Fluorescence(Ex=340 nm, Em=450 nm) was determined after a 5-minute incubation. Asummary of OPA data on all substrates is included in Table 3, above.

Soybean Trypsin Inhibitor Assays

Soybean Trypsin Inhibitor (SBTI, SIGMA-Aldrich, T6522) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialyzed, and incubatedwith SBTI (1 mg/ml final conc.) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis. Results are illustrated inTable 3, above. SDS-PAGE: for SBTI, following electrophoresis, proteinsin the gels were stained with Coomassie blue.

Wheat Germ Lectin assays

Wheat germ lectin (WGA, EY Laboratories, L-2101, Pure) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialysed, and incubatedwith WGA (1 mg/ml final concentration) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis as. Results are illustratedin Table 3, above. SDS-PAGE: for WGA, following electrophoresis,proteins in the gels were stained with Coomassie blue.

Soybean lectin assays

Soybean lectin (SBA, EY Laboratories, L-1300, Crude) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialysed, and incubatedwith SBA (1 mg/ml final concentration) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis. Results are illustrated inTable 3, above. SDS-PAGE: for SBA, following electrophoresis, proteinsin the gels were stained with Coomassie blue.

Gelatin in Fluorescent Liquid Endpoint Assay

DQ Gelatin (Molecular Probes, fluorescein conjugate, D-12054) was usedto assess the proteolytic activity of the proteases of the invention. DQgelatin is a protein that is so heavily labeled with a fluorophore thatits fluorescence is quenched when the molecule is intact. Proteases thatcleave the substrate will release the fluorophores from internalquenching and fluorescence will increase in proportion to the proteaseactivity. DQ Gelatin was diluted to a final concentration of 25 ug/ml in100 ul reactions containing a suitable buffer such as zymogramdeveloping buffer (Invitrogen) and varying amounts of proteasepreparations. Reactions were incubated in a 384 well, clear, flat-bottommicrotiter plate at 37° C. for various time periods from 1 hr toovernight. Fluorescence was monitored using a fluorescence plate readerafter incubation at 37° C. for various times.

As an example of the results obtained from the fluorescent liquid endpoint assay, see Table 5 and FIG. 5, which show the activity of SEQ IDNO:144 (encoded by SEQ ID NO:143). Samples were assayed in duplicate andthe raw data is shown in the Table 4, below. Duplicates were averagedand the background from the negative control was subtracted to depictthe increase in fluorescence caused by SEQ ID NO:144 activity in onehour using a bar graph (FIG. 5). TABLE 5 t = 0 t = 0 1 hour 1 hour SEQID NOS: 1759 1819 3660 3459 143, 144 Negative Control 1708 1785 18882069Peptides (Proteinase and Peptidase Activity)

Specificity Endpoint Assay

Synthetic small peptide substrates linked to a chromophore are oftenused to determine the specificity and aid in biochemicalcharacterization of proteases. To gauge the substrate specificity of theproteases of the invention, several para-nitroanalide linked syntheticpeptides were obtained from Sigma including Ala-Ala-Pro-Phe-pNA (AAPF),Ala-Ala-Ala-pNA (AAA), N-Bz-D,L-Arg-pNa (BAPNA), Gly-Gly-Phe-pNA,Ile-Glu-Gly-Arg-pNA, and Pro-Phe-Arg-pNA. When the peptide bond betweenthe pNA group and the amino acid in the P1 substrate position iscleaved, a yellow color is produced whose absorbance can be measured at410 nm. 25 mM stocks of small peptide substrates were prepared in DMSO.Substrates were used at a final concentration of 250 uM in 100 ulreaction volumes including varying amounts of protease preparations.Reactions were run in a suitable protease buffer such as 1× Zymogramdeveloping buffer from Invitrogen and were incubated in a 384 well,clear, flat-bottom microtiter plate at 37° C. for various time periodsfrom 1 hr to overnight. This “end point” assay provides a qualitativeinstead of quantitative method to assess substrate specificity. However,the process can be adapted to provide qualitative data by determininginitial rates for the various small peptide substrates.

Unit Definition Kinetic Assay

The following assay was developed to determine protease unit activityusing pNA linked small peptide substrates. This assay allows for thedirect comparison of enzymes of the invention to Subtilisin on a unitper unit basis. Free pNA was used to create a standard curve to allowconversion of pNA absorbance (A405 nm) to moles of pNA, allowing directquantification of the amount of pNA released by a protease (FIG. 6).

Subtilisin A activity (initial rate) on AAPF-pNA was measured over a 100fold concentration range of enzyme (0.1 to 10 U/mL in assay, based onSigma's supplied activity). The activity of Subtilisin A was linear withenzyme concentration over this range and allowed the determination ofequivalent units of enzymes of the invention over a broad activityrange. A Subtilisin A standard curve is shown in FIG. 7.

pH Assay

The following assay was developed using Subtilisin A to determine therelative activity of proteases at various pH's. Four different bufferswere identified that would permit the testing of a range of differentpH's. Protease activity was assayed using the small peptide substratep-nitroanalide linked Alanine-Alanine-Proline-Phenylalanine (AAPF-pNA,Sigma, S-7388) as follows: The amount of Subtilisin A required to obtainan initial rate using the assay conditions was determined at the desiredpH (5 mM AAPF-pNA, 37° C.). Reactions were performed in triplicate.Initial rates were determined and averaged. The percent activity atvarious pH's were determined relative to the sample with the highestactivity, and percent relative activity was then plotted vs. pH.Substrate stability at the pH's tested was verified in the absence ofactivity. Results are illustrated in Table 6 and in FIG. 8.

[see next page for Table 6] TABLE 6 Rates (A_(405nm) × 10³ min⁻¹) pHBuffer 1 2 3 Ave. Std Dev % Deviation % Relative Activity 5.0 Malic Acid3.71 3.80 3.62 3.71 0.09 2.5 10.09 5.5 Malic Acid 8.49 8.16 8.41 8.350.17 2.02 22.72 6.0 Malic Acid 13.56 13.24 12.23 13.01 0.69 5.32 35.385.5 MES 5.10 4.82 5.19 5.00 0.26 5.1 13.61 6.0 MES 11.81 11.53 11.1811.51 0.32 2.75 31.3 6.5 MES 20.45 19.48 20.49 20.14 0.57 2.85 54.76 7.0MES 27.54 27.51 27.03 27.36 0.28 1.03 74.41 6.5 MOPS 19.68 19.32 20.2019.73 0.44 2.24 53.66 7.0 MOPS 29.97 28.89 29.65 29.50 0.55 1.87 80.237.5 MOPS 34.24 34.02 32.65 33.64 0.86 2.55 91.47 8.0 MOPS 36.76 37.1936.37 36.77 0.41 1.12 100 8.0 Boric Acid 34.55 32.97 34.10 33.87 0.812.39 92.12 8.5 Boric Acid 35.39 32.01 35.41 34.27 1.96 5.72 93.19 9.0Boric Acid 34.85 33.99 33.45 34.10 0.70 2.07 92.72

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated or recombinant nucleic acid comprising a nucleic acidsequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreor complete sequence identity to SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5;SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ IDNO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ IDNO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ IDNO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ IDNO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ IDNO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ IDNO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ IDNO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ IDNO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ IDNO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125;SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ IDNO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164;SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ IDNO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254,over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150 or more residues, wherein the nucleic acid encodes at leastone polypeptide having a protease activity, and optionally the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection, and optionally the sequencecomparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall-p blastp-d “nr pataa”−F F, and allother options are set to default; (b) nucleic acid sequence encodes apolypeptide having a sequence as set forth in SEQ ID NO:2; SEQ ID NO:4;SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ IDNO:26; SEQ ID NO:28; SEQ ID NO:30: SEQ ID NO:32; SEQ ID NO:34; SEQ IDNO:36: SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44: SEQ IDNO:46: SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:52; SEQ ID NO:54; SEQ IDNO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ IDNO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ IDNO:76: SEQ ID NO:78; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ IDNO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94; SEQ IDNO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ ID NO:102; SEQ ID NO:104; SEQ IDNO:106; SEQ ID NO:108; SEQ ID NO:110; SEQ ID NO:112; SEQ ID NO:114; SEQID NO:116; SEQ ID NO:118; SEQ ID NO:120: SEQ ID NO:122; SEQ ID NO:124;SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO:132; SEQ IDNO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; SEQ ID NO:147; SEQ ID NO:151; SEQ ID NO:159; SEQ ID NO:165;SEQ ID NO:172; SEQ ID NO:180; SEQ ID NO:188; SEQ ID NO:194; SEQ IDNO:200; SEQ ID NO:205; SEQ ID NO:211; SEQ ID NO:219; SEQ ID NO:223; SEQID NO:230; SEQ ID NO:235; SEQ ID NO:242; SEQ ID NO:249 or SEQ ID NO:255;(c) a sequence that hybridizes under stringent conditions to a nucleicacid comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ IDNO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ IDNO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ IDNO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45: SEQ ID NO:47; SEQ IDNO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ IDNO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ IDNO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ IDNO:79; SEQ ID NO:81: SEQ ID NO:83; SEQ ID NO:85: SEQ ID NO:87: SEQ IDNO:89; SEQ ID NO:91: SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ IDNO:99; SEQ ID NO:101: SEQ ID NO:103: SEQ ID NO:105; SEQ ID NO:107; SEQID NO:109; SEQ ID NO:111; SEQ ID NO:113: SEQ ID NO:115; SEQ ID NO:117;SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ IDNO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ ID NO:135; SEQID NO:137; SEQ ID NO:139; SEQ ID NO:141: SEQ ID NO:143; SEQ ID NO:145;SEQ ID NO:146: SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164; SEQ IDNO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ ID NO:199; SEQID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQ ID NO:229;SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254, whereinthe nucleic acid encodes a polypeptide having a protease activity,wherein the stringent conditions comprise a wash step comprising a washin 0.2×SSC at a temperature of about 65° C. for about 15 minutes, andoptionally the nucleic acid is at least about 50, 75, 100, 150, 200,300, 400, 500, 600, 700, 800, 900, 1000 or more residues in length orthe full length of the gene or transcript; or (d) a sequencecomplementary to (a), (b) or (c); wherein optionally the proteaseactivity comprises catalyzing hydrolysis of peptide bonds; comprises anendoprotease activity or an exoprotease activity; comprises a proteinaseactivity or a peptidase activity; comprises a carboxypeptidase activity;comprises an aminopeptidase activity; comprises a serine proteaseactivity: comprises a metalloprotease activity, a matrix metalloproteaseactivity or a collagenase activity: comprises a cysteine proteaseactivity: comprises an aspartic protease activity: comprises achymotrypsin, a trypsin, an elastase, a kallikrein or a subtilisinactivity; or, comprises a dipeptidylpeptidase activity, whereinoptionally the protease activity is thermostable or thermotolerant.2-26. (canceled)
 27. A nucleic acid probe for identifying a nucleic acidencoding a polypeptide with a protease activity, wherein the probecomprises at least 10, or about 10 to 50, about 20 to 60, about 30 to70, about 40 to 80, about 60 to 100, or about 50 to 150, consecutivebases of (a) a sequence comprising SEQ ID NO:1; SEQ ID NO:3; SEQ IDNO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ IDNO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ IDNO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ IDNO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ IDNO:45; SEQ ID NO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ IDNO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ IDNO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ IDNO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ IDNO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ IDNO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ IDNO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123;SEQ ID NO:125; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ IDNO:133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQID NO:143; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158;SEQ ID NO:164; SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ IDNO:193; SEQ ID NO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQID NO:222; SEQ ID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 orSEQ ID NO:254, or (b) a sequence as set forth in claim 1; wherein theprobe identifies the nucleic acid by binding or hybridization understringent conditions, wherein the stringent conditions comprise a washstep comprising a wash in 0.2×SSC at a temperature of about 65° C. forabout 15 minutes. 28-32. (canceled)
 33. An amplification primer pair,wherein the primer pair comprises a first member having a sequence asset forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of (a) asequence as set forth in SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ IDNO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ IDNO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ IDNO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ IDNO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ IDNO:47; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:55; SEQ IDNO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ IDNO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ IDNO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ IDNO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:93; SEQ ID NO:95; SEQ IDNO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ IDNO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125;SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ ID NO:133; SEQ IDNO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQID NO:145; SEQ ID NO:146; SEQ ID NO:150; SEQ ID NO:158; SEQ ID NO:164;SEQ ID NO:171; SEQ ID NO:179; SEQ ID NO:187; SEQ ID NO:193; SEQ IDNO:199; SEQ ID NO:204; SEQ ID NO:210; SEQ ID NO:218; SEQ ID NO:222; SEQID NO:229; SEQ ID NO:234; SEQ ID NO:241; SEQ ID NO:248 or SEQ ID NO:254,or, (b) a sequence as set forth in claim 1; and a second member having asequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residuesof the complementary strand of the first member. 34-39. (canceled) 40.An expression cassette, a vector or a cloning vehicle comprising anucleic acid comprising a sequence as set forth in claim 1, whereinoptionally the cloning vehicle comprises a viral vector a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome, and optionally the viral vector comprises an adenovirusvector, a retroviral vector or an adeno-associated viral vector, andoptionally the cloning vehicle comprises a bacterial artificialchromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeastartificial chromosome (YAC), or a mammalian artificial chromosome (MAC).41-44. (canceled)
 45. A transformed cell comprising a nucleic acidcomprising a sequence as set forth in claim 1 or an expression cassetteexpression cassette, a vector or a cloning vehicle as set forth in claim40, wherein optionally the cell is a bacterial cell, a mammalian cell, afungal cell, a yeast cell, an insect cell or a plant cell. 46-47.(canceled)
 48. A transgenic non-human animal or transgenic plant ortransgenic seed comprising a sequence as set forth in claim 1 or anexpression cassette expression cassette, a vector or a cloning vehicleas set forth in claim 40 wherein optionally the animal is a mouse;wherein optionally the plant is a corn plant, a sorghum plant, a potatoplant, a tomato plant, a wheat plant, an oilseed plant, a rapeseedplant, a soybean plant, a rice plant, a barley plant, a grass, acottonseed, a palm, a sesame plant, a peanut plant, a sunflower plant ora tobacco plant: wherein optionally the seed is a corn seed, a wheatkernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, asunflower seed, a sesame seed, a rice, a barley, a peanut, a cottonseed,a palm, a peanut, a sesame seed, a sunflower seed or a tobacco plantseed. 49-56. (canceled)
 57. A double-stranded inhibitory RNA (RNAi)molecule comprising a subsequence of a sequence as set forth in claim 1wherein optionally the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more duplex nucleotides in length. 58-59. (canceled)
 60. Anisolated or recombinant polypeptide (i) having at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or has 100% sequence identity to SEQID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ IDNO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ IDNO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ IDNO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ IDNO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ IDNO:52; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ IDNO:62; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:68; SEQ ID NO:70; SEQ IDNO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:80; SEQ IDNO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ IDNO:92; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO:100; SEQ IDNO:102; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:108; SEQ ID NO:110; SEQID NO:112; SEQ ID NO:114; SEQ ID NO:116; SEQ ID NO:118; SEQ ID NO:120;SEQ ID NO:122; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:128; SEQ IDNO:130; SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQID NO:140; SEQ ID NO:142; SEQ ID NO:144; SEQ ID NO:147; SEQ ID NO:151;SEQ ID NO:159; SEQ ID NO:165; SEQ ID NO:172; SEQ ID NO:180; SEQ IDNO:188; SEQ ID NO:194; SEQ ID NO:200; SEQ ID NO:205; SEQ ID NO:211; SEQID NO:219; SEQ ID NO:223; SEQ ID NO:230; SEQ ID NO:235; SEQ ID NO:242;SEQ ID NO:249 or SEQ ID NO:255, or the polypeptide encoded by SEQ IDNO:145, over a region of at least about 50, 75, 100, 150, 200, 250, 300or more residues, wherein optionally the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection, or, (ii) encoded by a nucleic acid having a sequenceas set forth in claim 1 wherein optionally the polypeptide has aprotease activity, and optionally the protease activity comprisescatalyzing hydrolysis of peptide bonds; comprises an endoproteaseactivity or an exoprotease activity; comprises a proteinase activity ora peptidase activity; comprises a carboxypeptidase activity; comprisesan aminopeptidase activity; comprises a serine protease activity;comprises a metalloprotease activity; a matrix metalloprotease activityor a collagenase activity; comprises a cysteine protease activity;comprises an aspartic protease activity; comprises a chymotrypsin, atrypsin, an elastase, a kallikrein or a subtilisin activity; or,comprises a dipeptidylpeptidase activity, and optionally the proteaseactivity is thermostable or thermotolerant; and optionally thepolypeptide comprises at least one glycosylation site, whereinoptionally the glycosylation is an N-linked glycosylation: andoptionally the polypeptide retains a protease activity under conditionscomprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0. andoptionally the polypeptide retains a protease activity under conditionscomprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5.61-97. (canceled)
 98. An array comprising an immobilized polypeptide asset forth in claim 60, or an immobilized nucleic acid as set forth inclaim 1; wherein optionally the polypeptide or nucleic acid isimmobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass,a microelectrode, a graphitic particle, a bead, a gel, a plate, an arrayor a capillary tube.
 99. (canceled)
 100. An isolated or recombinantantibody that specifically binds to a polypeptide as set forth in claim60 or encoded by a nucleic acid as set forth in claim 1; whereinoptionally the antibody is a monoclonal or a polyclonal antibody.101-105. (canceled)
 106. A method of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid operably linked toa promoter, wherein the nucleic acid comprises a sequence as set forthin claim 1; and (b) expressing the nucleic acid of step (a) underconditions that allow expression of the polypeptide, thereby producing arecombinant polypeptide. 107-115. (canceled)
 116. A computer systemcomprising a processor and a data storage device, or a computer readablemedium, wherein said data storage device or a computer readable mediumhas stored thereon a polypeptide sequence or a nucleic acid sequence,wherein the polypeptide sequence comprises sequence as set forth inclaim 60, a polypeptide encoded by a nucleic acid as set forth in claim1, wherein optionally the system further comprises a sequence comparisonalgorithm, and optionally the sequence comparison algorithm comprises acomputer program that indicates polymorphisms, and optionally the systemfurther comprises an identifier that identifies one or more features insaid sequence. 117-125. (canceled)
 126. A method for isolating orrecovering a nucleic acid encoding a polypeptide with a proteaseactivity from an environmental sample comprising the steps of: (a)providing an amplification primer sequence pair as set forth in claim 33or a polynucleotide probe comprising a sequence as set forth in claim27; (b) isolating a nucleic acid from the environmental sample ortreating the environmental sample such that nucleic acid in the sampleis accessible for hybridization to the amplification primer pair orprobe; and, (c) combining the nucleic acid of step (b) with theamplification primer pair or probe of step (a) and amplifying oridentifying a nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide with aprotease activity from an environmental sample, and optionally theenvironmental sample comprises a water sample, a liquid sample, a soilsample an air sample or a biological sample; and optionally thebiological sample is derived from a bacterial cell, a protozoan cell, aninsect cell, a yeast cell, a plant cell, a fungal cell or a mammaliancell. 127-130. (canceled)
 131. A method of generating a variant of anucleic acid encoding a polypeptide with a protease activity comprisingthe steps of: (a) providing a template nucleic acid comprising asequence as set forth in claim 1; and (b) modifying, deleting or addingone or more nucleotides in the template sequence, or a combinationthereof, to generate a variant of the template nucleic acid; optionallyfurther comprising expressing the variant nucleic acid to generate avariant protease polypeptide: optionally wherein the modifications,additions or deletions are introduced by a method comprising error-pronePCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCRsexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,recursive ensemble mutagenesis, exponential ensemble mutagenesis,site-specific mutagenesis, gene reassembly, gene site saturatedmutagenesis (GSSM), synthetic ligation reassembly (SLR) and acombination thereof: optionally wherein the modifications, additions ordeletions are introduced by a method comprising recombination, recursivesequence recombination, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesischemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof; optionally the method isiteratively repeated until a protease having an altered or differentactivity or an altered or different stability from that of a polypeptideencoded by the template nucleic acid is produced, and optionally thealtered or different activity protease polypeptide is thermotolerant,and retains some activity after being exposed to an elevatedtemperature; optionally the method is iteratively repeated until aprotease coding sequence having an altered codon usage from that of thetemplate nucleic acid is produced: optionally the method is iterativelyrepeated until a protease gene having higher or lower level of messageexpression or stability from that of the template nucleic acid isproduced. 132-140. (canceled)
 141. A method for modifying codons in anucleic acid encoding a polypeptide with a protease activity to increaseits expression in a host cell, the method comprising the followingsteps: (a) providing a nucleic acid encoding a polypeptide with aprotease activity comprising a sequence as set forth in claim 1; and,(b) identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell,or, identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding a protease,or, identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.142-172. (canceled)
 173. A method for hydrolyzing, breaking up ordisrupting a protein-comprising composition comprising the followingsteps: (a) providing a polypeptide having a protease activity as setforth in claim 60; (b) providing a composition comprising a protein; and(c) contacting the polypeptide of step (a) with the composition of step(b) under conditions wherein the protease hydrolyzes, breaks up ordisrupts the protein-comprising composition; optionally wherein thecomposition comprises a plant cell, a bacterial cell, a yeast cell, aninsect cell, or an animal cell.
 174. A method for liquefying or removinga protein from a composition comprising the following steps: (a)providing a polypeptide having a protease activity as set forth in claim60; (b) providing a composition comprising a protein; and (c) contactingthe polypeptide of step (a) with the composition of step (b) underconditions wherein the protease removes or liquefies the protein.
 175. Adetergent composition comprising a polypeptide as set forth in claim 60,wherein the polypeptide has a protease activity, wherein optionally theprotease is a non-surface-active protease or a surface-active protease,and optionally the protease is formulated in a non-aqueous liquidcomposition, a cast solid, a granular form, a particulate form, acompressed tablet, a gel form, a paste or a slurry form. 176-179.(canceled)
 180. A textile or fabric comprising a polypeptide as setforth in claim 60, wherein optionally the textile or fabric comprises acellulose-containing fiber. 181-184. (canceled)
 185. A feed or a foodcomprising a polypeptide as set forth in claim
 60. 186-189. (canceled)190. A dairy product comprising a protease having a sequence as setforth in claim
 60. 191-195. (canceled)
 196. A paper or paper product orpaper pulp comprising a protease as set forth in claim
 60. 197.(canceled)
 198. A pharmaceutical composition comprising a polypeptidehaving protease activity as set forth in claim 60, wherein optionallythe pharmaceutical composition acts as a digestive aid or as a topicalskin care, or an antimicrobial, antiviral or an antitoxin agent, or ananticancer agent, wherein optionally the polypeptide having proteaseactivity is present in a therapeutically effective amount to cleave anddestroy a specific target protein, wherein optionally the target proteincomprises a toxin protein, an essential viral or a cancer cell protein,wherein optionally the toxin protein is an Anthrax toxin, a Clostridiumbotulinum toxin, or a Ricin toxin. 199-201. (canceled)
 202. An oral careproduct comprising a polypeptide as set forth in claim 60, whereinoptionally the product comprises a toothpaste, a dental cream, a gel ora tooth powder, an odontic, a mouth wash, a pre- or post-brushing rinseformulation, a chewing gum, a lozenge or a candy.
 203. (canceled)
 204. Acontact lens cleaning composition comprising a polypeptide as set forthin claim
 60. 205. A method for treating solid or liquid animal wasteproducts comprising the following steps: (a) providing a polypeptide asset forth in claim 60; (b) providing a solid or a liquid animal waste;and (c) contacting the polypeptide of step (a) and the solid or liquidwaste of step (b) under conditions wherein the protease can treat thewaste.
 206. A processed waste product comprising a polypeptide having aprotease activity, wherein the polypeptide comprises a sequence as setforth in claim
 60. 207-211. (canceled)
 212. An antimicrobial, anti-viralor anti-spore agent comprising a polypeptide having a protease activity,wherein the polypeptide comprises a sequence as set forth in claim 60,wherein optionally the protease has antimicrobial or anti-viral activitycomprising hydrolysis of a protein, wherein optionally the proteincomprises an Anthrax toxin, Clostridium botulinum toxin or a Ricintoxin.
 213. A disinfectant comprising a polypeptide having a proteaseactivity, wherein the polypeptide comprises a sequence as set forth inclaim
 60. 214. A method for tissue dissociation comprising the followingsteps: (a) providing a composition comprising a polypeptide having aprotease activity as set forth in claim 60; and (b) contacting thecomposition of step (a) and with a tissue to be dissociated, whereinoptionally the tissue is a wounded tissue and optionally the contactingof step (b) is used for wound cleansing, wound bed preparation to treatpressure ulcers, leg ulcers, burns diabetic foot ulcers, scars I.V.fixation, surgical wounds or minor wounds. 215-217. (canceled)
 218. Amedical dressing comprising a polypeptide having a protease activity,wherein the polypeptide comprises a sequence as set forth in claim 60.219. An antitoxin composition comprising a polypeptide having a proteaseactivity comprising the ability to hydrolyze a toxin, wherein thepolypeptide comprises a sequence as set forth in claim 60, whereinoptionally the toxin comprises Anthrax toxin, Clostridium botulinumtoxin, Ricin toxin.
 220. A decontamination or anti-biological warfareagent comprising a polypeptide having a protease activity as set forthin claim 60, wherein optionally the polypeptide having a proteaseactivity has the ability to hydrolyze a toxin or an essential viral ormicrobial agent.
 221. A medicament or pharmaceutical formulated for usein tissue dissociation, wound cleansing, wound bed preparation, treatingpressure ulcers, treating leg ulcers, treating burns, treating diabeticfoot treating ulcers, treating scars, intravenous (I.V.) fixation, ortreating surgical wounds or minor wounds, wherein the medicament orpharmaceutical comprises a polypeptide having a protease activity as setforth in claim 60, wherein optionally the tissue is a wound.
 222. Use ofa composition in the manufacture of a medicament or pharmaceutical fortissue dissociation, wound cleansing, wound bed preparation, treatingpressure ulcers, treating leg ulcers, treating burns, treating diabeticfoot treating ulcers, treating scars, intravenous (I.V.) fixation, ortreating surgical wounds or minor wounds, wherein the compositioncomprises a polypeptide having a protease activity as set forth in claim60, wherein optionally the tissue is a wound, or optionally thepharmaceutical is formulated for wound cleansing, wound bed preparation,to treat pressure ulcers, leg ulcers, burns, diabetic foot ulcers,scars, intravenous (I.V.) fixation, surgical wounds or minor wounds.